Yesterday one of the post-docs and I discussed whether we should submit a proposal to NIH. Yes, we did just get one proposal funded, to work on the regulation of competence genes in H. influenzae and E. coli. But her project is completely different, and some parts of it are going to be expensive.
She's studying when, how and why different lineages of H. influenzae lose (or maybe gain) the ability to take up DNA and recombine it into the chromosome. We already knew that this occurs in various bacteria, and she's now completing a thorough analysis of the variation in DNA uptake and recombination ability in a broad selection of H. influenzae strains.
Some of these strains were chosen because their genomes have been or are in the process of being completely sequenced (one of the benefits of working on a sometime pathogen), and her next goal is to analyze these sequences for differences that could explain their different phenotypes. This bioinformatics work won't cost much except her time; we've already bought a nice fast computer for it. And her time doesn't cost the lab anything, because she's supported by a lovely post-doctoral fellowship from NIH.
But the next steps will be expensive. She wants to use the bioinformatics information to design investigations into the genetic differences of strains that haven't been sequenced. Her original plan was to develop a microarray chip containing all the genes and alleles that the bioinformatics and other work suggested might be involved. This still seems like a good approach, but the field is changing so fast that better ways to survey genomes are becoming available faster than we can keep up. One thing they have in common is that they'll all cost a bundle.
Subsequent work will also be pricey. We'll probably want to follow up the H. influenzae findings with investigation into related bacteria. This will be beyond the scope of the present post-doc, so we'll need new post-docs or grad students or technicians, as well as money for the tools and techniques.
The other reason to write a proposal to NIH is that proposal-writing is the best framework I know of for clear thinking about research plans.
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Showing posts with label bacterial competence. Show all posts
Showing posts with label bacterial competence. Show all posts
Polishing the Sxy manuscript
We're fixing the final details on the Sxy manuscript; I'm hoping to have it submitted in the next 24 hours. Below I'll try to summarize what it says, in less technical terms than the Abstract uses.
We already know that the Sxy protein regulates expression of competence genes; here we're examining how Sxy itself is regulated. Our most powerful tools are regulatory mutations that turn Sxy on when it would otherwise be off. The paper starts by describing new mutations that, taken all together, strongly suggest that expression is controlled by changes in RNA folding. We conclude this because all of the mutations change how RNA can fold, but only one of them changes the Sxy protein sequence (and that in a trivial way).
The paper then presents data showing that the RNA folding changes don't affect how much Sxy RNA is made, but how efficiently the RNA is used to make Sxy protein. Then more data shows that normal cells translate their Sxy RNA more efficiently when they're starved, but the mutant cells translate it efficiently even when they're not starved.
The cell uses the genes/proteins that Sxy regulates to take up DNA, so these results help us understand how being starved causes cells to take up DNA. We have argued that cells take up DNA as food (not to get different versions of their genes as others have assumed), so these results strengthen the evidence that DNA uptake is an adaptive response to starvation.
We already know that the Sxy protein regulates expression of competence genes; here we're examining how Sxy itself is regulated. Our most powerful tools are regulatory mutations that turn Sxy on when it would otherwise be off. The paper starts by describing new mutations that, taken all together, strongly suggest that expression is controlled by changes in RNA folding. We conclude this because all of the mutations change how RNA can fold, but only one of them changes the Sxy protein sequence (and that in a trivial way).
The paper then presents data showing that the RNA folding changes don't affect how much Sxy RNA is made, but how efficiently the RNA is used to make Sxy protein. Then more data shows that normal cells translate their Sxy RNA more efficiently when they're starved, but the mutant cells translate it efficiently even when they're not starved.
The cell uses the genes/proteins that Sxy regulates to take up DNA, so these results help us understand how being starved causes cells to take up DNA. We have argued that cells take up DNA as food (not to get different versions of their genes as others have assumed), so these results strengthen the evidence that DNA uptake is an adaptive response to starvation.
The controversy surrounding the function of DNA uptake
I'm holed up in Indio California, in a "RV Resort" for retirees, working on my grant proposal and checking out the local attractions (Washingtonia palms! The Salton Sea!).
Here's a few paragraphs from the proposal introduction, explaining the big question:
The consequences of DNA uptake are not in question. A cell that takes up DNA inevitably incurs the physiological costs of becoming competent and of transporting the DNA across its envelope. The cell also inevitably gets the incoming DNA’s nucleotides, reducing the demands on its biosynthetic or salvage pathways. Because DNA is abundant in natural environments, and nucleotides are very expensive to synthesize, the nucleotide benefit may be sufficient to compensate for the costs and thus to explain the evolution (origin and continuation) of competence. However if the incoming DNA recombines with the chromosome it may also change the cell’s genotype, which may increase or decrease the cell’s ability to survive and reproduce. The controversial question is whether natural selection on the machinery and regulation of DNA uptake has been influenced by these genetic consequences.
The conventional view is that bacteria take up DNA for recombination (i.e., that recombination has net benefits, and that these are sufficient to account for the evolution of competence). This derives partly from the now-discredited idea that sex in eukaryotes is easily explained by long-term benefits to the species, and partly from observation of ancient beneficial recombination events in bacterial genomes and recent ones in the laboratory. But there are also substantial costs to genetic recombination, because the homologous DNA in the environment comes from dead cells and is likely to carry excess deleterious mutations, and because recombination with heterologous DNA will usually the cell’s well-adapted genetic machinery. These genetic costs are easily overlooked because natural selection eliminates the cells incurring them.
Understanding the evolution of bacterial competence has major implications for our present far-from-satisfactory understanding of why sexual reproduction evolved in eukaryotes. The problematic hypothesis that meiotic sex evolved to create new combinations of genes is often supported by claims that bacterial ‘parasexual’ processes also evolved for this. Because conjugation and transduction are now known to be side effects of selection for more immediate benefits to cells or their genetic parasites, understanding competence is key. If the genetic consequences of competence have not shaped its mechanism or regulation, we will conclude that bacteria get all the genetic variation they need by accident, and thus that meiotic sex is a eukaryotic solution to a eukaryotic problem.
Direct experimental testing of proposed costs and benefits is not the best approach, because it is all too easy to create selection in bacterial cultures, and because laboratory conditions in no way replicate those of the natural environment. Rather, the best way to understand the evolution of competence is to understand its regulation and mechanism. Regulation is informative for all bacteria, as the genes that regulate competence evolved in the natural environment, and understanding the signals they respond to will give us a window on the consequences of competence that have been most beneficial. Because H. influenzae’s uptake specificity causes it to preferentially take up its own DNA, understanding the uptake mechanism responsible for this bias is a critical test of the importance of recombination.
Here's a few paragraphs from the proposal introduction, explaining the big question:
The consequences of DNA uptake are not in question. A cell that takes up DNA inevitably incurs the physiological costs of becoming competent and of transporting the DNA across its envelope. The cell also inevitably gets the incoming DNA’s nucleotides, reducing the demands on its biosynthetic or salvage pathways. Because DNA is abundant in natural environments, and nucleotides are very expensive to synthesize, the nucleotide benefit may be sufficient to compensate for the costs and thus to explain the evolution (origin and continuation) of competence. However if the incoming DNA recombines with the chromosome it may also change the cell’s genotype, which may increase or decrease the cell’s ability to survive and reproduce. The controversial question is whether natural selection on the machinery and regulation of DNA uptake has been influenced by these genetic consequences.
The conventional view is that bacteria take up DNA for recombination (i.e., that recombination has net benefits, and that these are sufficient to account for the evolution of competence). This derives partly from the now-discredited idea that sex in eukaryotes is easily explained by long-term benefits to the species, and partly from observation of ancient beneficial recombination events in bacterial genomes and recent ones in the laboratory. But there are also substantial costs to genetic recombination, because the homologous DNA in the environment comes from dead cells and is likely to carry excess deleterious mutations, and because recombination with heterologous DNA will usually the cell’s well-adapted genetic machinery. These genetic costs are easily overlooked because natural selection eliminates the cells incurring them.
Understanding the evolution of bacterial competence has major implications for our present far-from-satisfactory understanding of why sexual reproduction evolved in eukaryotes. The problematic hypothesis that meiotic sex evolved to create new combinations of genes is often supported by claims that bacterial ‘parasexual’ processes also evolved for this. Because conjugation and transduction are now known to be side effects of selection for more immediate benefits to cells or their genetic parasites, understanding competence is key. If the genetic consequences of competence have not shaped its mechanism or regulation, we will conclude that bacteria get all the genetic variation they need by accident, and thus that meiotic sex is a eukaryotic solution to a eukaryotic problem.
Direct experimental testing of proposed costs and benefits is not the best approach, because it is all too easy to create selection in bacterial cultures, and because laboratory conditions in no way replicate those of the natural environment. Rather, the best way to understand the evolution of competence is to understand its regulation and mechanism. Regulation is informative for all bacteria, as the genes that regulate competence evolved in the natural environment, and understanding the signals they respond to will give us a window on the consequences of competence that have been most beneficial. Because H. influenzae’s uptake specificity causes it to preferentially take up its own DNA, understanding the uptake mechanism responsible for this bias is a critical test of the importance of recombination.
Progress on the sxy manuscript
I've been working on the sxy manuscript, clarifying/rewriting the text and improving the figures. I'm not yet all the way through the Results but it's looking a lot better.
The Introduction wasn't too bad anyway, but now it flows better and explains the issues in a more logical order. It's also 65 words shorter, mostly because I cut out some repetition and tightened the sentences.
The Results section is a lot better now. I polished Figure 2, which shows the structure of the sxy gene's 5' end, and now point out that this untranslated region is unusually long. The correlation between Sxy protein and competence is stated rather than just being implied, and backed up with Figure 4. We have a beautiful new version of Figure 5, with all the bands clearly visible, but the text describing these results still needs work. The description of the compensatory mutation work is clearer.
I came up with a 'spin' that turns a discrepancy into a result: RNase analysis shows little difference between mutant and wild-type sxy structures, but the phenotypes are very different. I now say this means that the structure is stable in vitro but labile in vivo, probably due to dynamic effects while the mRNA is being synthesized.
My explanation of the fusions of sxy to lacZ are now much clearer, mainly because my many attempts to really understand this data finally paid off. I've inverted the order in which we discuss the normal (long) and stem-free (short) fusions. The short ones were created as a bit of an afterthought (no, that's probably unfair to the grad student who did this - these fusions we something she thought up herself, and I don't know when). Anyway, I realized that we should consider them first, and then contrast them with the fusions that have the normal structure. I think it's much easier to think about this way. And my new clarity also produced a newly-clear figure, with simple schematic drawings of the fusion structures.
And then my brain shut down for the day.
The Introduction wasn't too bad anyway, but now it flows better and explains the issues in a more logical order. It's also 65 words shorter, mostly because I cut out some repetition and tightened the sentences.
The Results section is a lot better now. I polished Figure 2, which shows the structure of the sxy gene's 5' end, and now point out that this untranslated region is unusually long. The correlation between Sxy protein and competence is stated rather than just being implied, and backed up with Figure 4. We have a beautiful new version of Figure 5, with all the bands clearly visible, but the text describing these results still needs work. The description of the compensatory mutation work is clearer.
I came up with a 'spin' that turns a discrepancy into a result: RNase analysis shows little difference between mutant and wild-type sxy structures, but the phenotypes are very different. I now say this means that the structure is stable in vitro but labile in vivo, probably due to dynamic effects while the mRNA is being synthesized.
My explanation of the fusions of sxy to lacZ are now much clearer, mainly because my many attempts to really understand this data finally paid off. I've inverted the order in which we discuss the normal (long) and stem-free (short) fusions. The short ones were created as a bit of an afterthought (no, that's probably unfair to the grad student who did this - these fusions we something she thought up herself, and I don't know when). Anyway, I realized that we should consider them first, and then contrast them with the fusions that have the normal structure. I think it's much easier to think about this way. And my new clarity also produced a newly-clear figure, with simple schematic drawings of the fusion structures.
And then my brain shut down for the day.
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