If we decide to follow the CIHR proposal outline I've made in the previous post, we'll probably do a separate proposal to NSERC on the role of uptake sequences in the mechanism of DNA uptake. I was just discussing this with the post-doc and he had a great idea about also using the analysis to make inferences about the frequencies of chromosomal recombination in naturally competent species. So I'm going to write it out here quickly, before I forget it.
Motivation: This work addresses a very important question with big/deep/fundamental importance to the colossal problem of the origin of meiotic sex in eukaryotes. The question is 'Do bacteria have any processes that evolved because of selection for recombination of chromosomal alleles?' We think this selection is the reason for the success of meiotic sexual reproduction in eukaryotes, but compelling evidence for this has been elusive. Bacteria have four well-studied processes that do generate homologous recombination; three that transfer DNA between cells and one that carries out homologous recombination. But almost every aspect of these processes has been shown to cause recombination as an unselected side effect of processes selected for other functions.
Natural competence, the ability to take up DNA fragments from the environment, is the only one of the four for which reasonable doubt remains about its non-recombinational function. The strongest selection for DNA uptake is generated by its nutritional consequences; DNA is an excellent and economical source of preformed nucleotides and of phosphate, and the nutrient function of DNA uptake is supported by its regulation by nutritional signals in many bacteria. This doubt arises from the apparent self-specificity of DNA uptake by bacteria in two groups, the genus Neisseria and the family Pasteurellaceae. Bacteria in these groups preferentially take up DNAs containing short sequence motifs that are ~100-fold more abundant in their own genomes than expected by chance. This match between the bias of the DNA uptake machinery and the genomic abundance of a DNA motif has been interpreted as an adaptation that enhances the presumed recombinational (sexual) benefits of DNA uptake, by allowing mate-choice or excluding possibly harmful foreign DNAs. However a simpler non-sexual explanation exists - that the preferred sequences play purely mechanistic roles in DNA uptake, and that the motifs' abundances in the respective genomes are due to a passive accumulation caused by biased uptake and subsequent unselected recombination. (Something here about why mechanistic sequence biases are plausible/expected.)
Only a subset of competent species exhibit this uptake specificity (strongly biased uptake and strongly enriched genome). The others are considered to have no uptake bias at all (and no genomic uptake sequences), largely because they show no preference for DNA fragments form their own genomes over those from other genomes. Thus the most powerful test of this hypothesis is testing these species for cryptic uptake biases. Finding of even minor biases in their uptake machinery would confirm that biased uptake need not result from selection for mate-choice. We have developed a simple and very powerful method to test for such biases, using Illumina sequencing of DNA fragments taken up from pools of highly degenerate DNA fragments.
As a bonus, this analysis will allow estimation, for a number of bacterial species, of the frequencies of chromosomal recombination between close relatives. We will be working with species whose genomes have been sequenced. Once any uptake biases have been identified, the corresponding genomes can be analyzed to detect any enrichment of the preferred motifs. (Unless the motifs are as complex as the known Neisserial and Pasteurellacean ones) For motifs that are either short or not very strong, finding enrichment will not be evidence for a sexual function, since the same motifs will occur frequently in foreign DNAs. However the enrichment will allow estimation of the frequency of recombination in the species, because we have developed a computer-simulation model of the accumulation process.
A. Test naturally competent bacteria for biases in their DNA uptake mechanisms. We've done H. influenzae, which has well characterized uptake bias. We'll test Acinetobacter bayleyi and Thermus thermophilus, both Gram-negative bacteria thought to have no uptake bias and no genomic enrichment. I've already arranged collaborations with a researcher; it will probably be simplest to travel to their lab to do the uptake experiments. We'll use fully degenerate Illumina-ready DNA fragments, which we have already designed and ordered. The analysis methods for the Illumina (or Miseq) output have already been developed for H. influenzae; they may need modification for the fully degenerate sequences. We could also test a Vibrio (maybe not cholerae!) and maybe Pseudomonas stutzeri. We could also test bacteria that have some uptake bias, with (A. pleuropneumoniae and G. anatis) or without (Campylobacter) genomic uptake motifs, but this wouldn't really test our hypothesis.
Could we also test Gram-positive bacteria for uptake bias (Streptococcus or Bacillus)? This is the equivalent of testing Gram-negative bacteria for translocation bias, and will be substantially more difficult because as the DNA enters the cytoplasm it becomes single-stranded and partly degraded (at one end). But we think we can develop ways to fish out the single strands from the cytoplasm.
What if we find no evidence of bias? I'd be surprised. This wouldn't disprove our hypothesis, but it would weaken it, because our hypothesis is built on the expectation that all DNA binding proteins have some sequence specificity, and that tasks requiring application of force to DNA will benefit from the tighter binding created by sequence-specific interactions.
B. Examine the corresponding genome sequences for overrepresentation of the preferred motifs. If we find no overrepresentation, we will have established that uptake biases need not result from selection for preferential uptake of self-DNA. If we do find overrepresentation, we can (1) decide whether this overrepresentation would create any significant degree of preferential uptake of self-DNA, and (2) use our simulation model (or refinements of it) to evaluate how much recombination must be going on to give this overrepresentation.
Back to the big picture: If we find uptake bias without genomic overrepresentation, or without self-specificity, we'll have swept away the last feeble pillar still propping up the claim that bacteria have processes that evolved for genetic exchange.
13 hours ago in Variety of Life