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.
Aims:
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.
Last time I checked, no one knows even how heat-shock transformation of E.coli works. Seems like there was an active research on the subject in the 1970s and 1980s but it proved too difficult, and so it fizzled as not worthy of attention.
ReplyDeleteI haven't actually followed the literature for something like 20 years but back then the field that studies bacterial natural transformation competence and mechanisms of DNA uptake was in embryonic state with hardly anything understood. Whether there is DNA sequence bias is, IMO, not even a particularly interesting question on the background of the question of how a highly hydrophilic molecule gets of a size comparable with cell size gets through the cell wall and hydrophobic plasma membrane.
Hi DK: If you'd like to bring your knowledge up to date you could read our latest grant proposal. It's here.
ReplyDeleteI looked at it (obviously, not detailed enough to check out refs and all that but enough to get a sense of what's there). Just IMO: I find the emphasis on proteins really annoying and misguided. There is a fundamental physical problem and you are trying to reduce it to what seems to be a small and not particularly representative subset of related phenomena. To illustrate with analogy: to understand how ribosome works, one needs to figure that it's RNA doing most of the work. Focusing on its proteins may get you funded but won't get you very far with regard to understanding of the what's going on.
ReplyDeleteP.S. Interesting to see that not much has changed in 20 years.
But in the case of DNA uptake, the proteins ARE what's doing the work. This is active uptake, not passive permeation as in the E. coli 'heat shock' transformation you referred to.
ReplyDeleteYou're right that the DNA properties do matter. We're also analyzing the physical properties of the DNA fragments that are preferentially taken up, as we hypothesize that these properties play a big role in the initiation of uptake. And we're also going to look for sequence effects in the post-initiation progression of uptake.
But in the case of DNA uptake, the proteins ARE what's doing the work. This is active uptake, not passive permeation as in the E. coli 'heat shock' transformation you referred to.
ReplyDeleteSince I don't think you know the mechanism of either, I don't see what makes you so confident making such statements. Personally, I'd bet that you are wrong and that the "active uptake" is merely a protein-assisted version of the more general phenomenon.
I'm not wasting any more courtesy on an ignorant troll...
ReplyDeleteGood retort! Very effective and to the point! A minor thing remains - tell the actual mechanisms then...
ReplyDeleteBut, just so you may understand my point a little better, another analogy: There is no doubt that proteins play a crucial role in membrane fusion. At the same time, by now it is 100% clear that all they do is promote the basic sequence of events that are observed without any proteins in many "artificial" membrane fusions (PEG-, osmotic shock-, peptite-mediated, etc).
What do you think about gene transfer agents? They don't appear to me to have any obvious function besides transferring bacterial DNA. Clearly they evolved from phages that didn't care one bit about transferring anything but their genome, but they don't do that any more. And they seem to be under purifying selection.
ReplyDeleteI really don't know what to make of them. I suspect they're selected on because they have direct effects on the cells, independent of the gene transfer they cause. Being a donor apparently requires cell death, which makes selection for transfer effects particularly unlikely.
Delete