The postdoc and I have finished making revisions to his first manuscript (about recombination tracts, provisionally accepted by PLoS Pathogens). He made a beautiful new figure summarizing all the data, and then did a very nice new analysis of sequence variation between the donor and recipient DNAs, which he turned into another lovely new figure. Then he rewrote bits of the Results, and we rewrote most of the Discussion, with a lot of back and forth and some squabbling. But now it's just about perfect, though he may be doing some (I hope tiny) final edits before resubmitting it.
He's out of town for a couple of weeks, working with his collaborator, and I'm turning my attention to his second manuscript (the one about uptake specificity). We had originally hoped to get it submitted when our current CIHR grant proposal went in at the start of March, but it was far from ready. And in the interim he has come up with better ways to analyze the data. He wants to put off paper-writing until this new analysis is complete, but I think we should try to get the manuscript as close to finished as possible while the new data is being generated. So I'm reading the very rough draft he gave me a few weeks ago, and getting ideas of what's needed.
Field of Science
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From Valley Forge to the Lab: Parallels between Washington's Maneuvers and Drug Development4 weeks ago in The Curious Wavefunction
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Political pollsters are pretending they know what's happening. They don't.4 weeks ago in Genomics, Medicine, and Pseudoscience
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Course Corrections5 months ago in Angry by Choice
Rethinking the nucleases (and DprA and ComM)
So maybe the DprA and ComM proteins don't directly protect DNA from nucleases, at least not in Haemophilus. Maybe instead they do something (or two different somethings) that promote homologous recombination, and the recombination is just the normal consequence of not recombining.
But in S. pneumoniae, I think there's good evidence that DNA that can't recombine with the chromosome degrades faster in a dprA-knockout strain. Better check... OK, an excellent 2003 paper by Berge et al. showed that incoming plasmid DNA with no homology to the chromosome is rapidly degraded in both recA and dprA mutants. The degradation is faster than the normal kinetics of recombination in wildtype cells, so it isn't just a consequence of the lack of recombination.
Back to the roles of specific nucleases. Something certainly degrades the DNA, and the control transformations of our nuclease experiments confirm that it isn't ExoV or ExoI or RecJ, because each nuclease mutation reduces the transformation frequency. (We can't even claim originality on this conclusion, because the researchers who made the nuclease mutants also showed this.)
Why do I care what these two proteins do? Both have been interpreted as evidence that the selected function of DNA uptake is to generate recombinants. I think this is incorrect because of the larger picture view of selection for recombination, not because of any flaw in the experiments. The big question is, what do these proteins do for cells in the absence of DNA uptake.
To a first approximation, the dprA and comM mutants show normal viability and growth and sensitivity to UV. The E. coli DprA paper showed that E. coli DprA can partially complement the transformation defect of an H. influenzae knockout, which the authors interpreted as meaning that DprA's function in non-competent cells also contributes to transformation in cells that take up DNA.
But in S. pneumoniae, I think there's good evidence that DNA that can't recombine with the chromosome degrades faster in a dprA-knockout strain. Better check... OK, an excellent 2003 paper by Berge et al. showed that incoming plasmid DNA with no homology to the chromosome is rapidly degraded in both recA and dprA mutants. The degradation is faster than the normal kinetics of recombination in wildtype cells, so it isn't just a consequence of the lack of recombination.
Back to the roles of specific nucleases. Something certainly degrades the DNA, and the control transformations of our nuclease experiments confirm that it isn't ExoV or ExoI or RecJ, because each nuclease mutation reduces the transformation frequency. (We can't even claim originality on this conclusion, because the researchers who made the nuclease mutants also showed this.)
Why do I care what these two proteins do? Both have been interpreted as evidence that the selected function of DNA uptake is to generate recombinants. I think this is incorrect because of the larger picture view of selection for recombination, not because of any flaw in the experiments. The big question is, what do these proteins do for cells in the absence of DNA uptake.
To a first approximation, the dprA and comM mutants show normal viability and growth and sensitivity to UV. The E. coli DprA paper showed that E. coli DprA can partially complement the transformation defect of an H. influenzae knockout, which the authors interpreted as meaning that DprA's function in non-competent cells also contributes to transformation in cells that take up DNA.
The grad student's research
I met with the grad student this morning and we went over his data. His experiments aren't clean (he's still learning to slow down and think carefully about what he's going to do, and to keep careful thorough notes), but they provide a strong indication that the ComM protein's function is not to protect DNA from the RecJ and ExoI nucleases. The undergrad who worked on this before him showed the same thing for DprA, and the undergrad before that showed it for ComM and ExoV and I showed it before that for DprA and ExoV.
So here's a plan: First we write out an outline of the paper we hope to publish about these results. What will this paper say? First, it will summarize the evidence that DprA and ComM protect DNA from degradation. For DprA this has been done thoroughly for Streptococcus pneumoniae; in other bacteria the dprA knockout phenotype is consistent with this but I don't know if DNA degradation has been directly measured. But the comM gene isn't present in other competent bacteria, so I think we might first need to show that it does indeed protect incoming DNA against nuclease attack.
Hmmm, I just looked at the 1998 paper describing the discovery of comM and characterization of the mutant phenotype (Gwinn et al. 1998. J. Bacteriol. 180:746-748). They did translocation assays (figure below) where cells were given linearized end-labeled DNA - one fragment cloned from the chromosome*, one vector fragment. The fragments were degraded, and the radioactivity from both fragments wound up in the chromosome, but not any faster in the comM mutant than in wildtype cells. This doesn't provide any support for my assumption that ComM protects DNA from degradation! The rec2 and comF mutants are controls, blocking translocation of DNA into the cytoplasm, and the rec1 mutation blocks homologous recombination.
So, if ComM (maybe) doesn't protect DNA from degradation, what else might it be doing that promotes recombination? And maybe DprA too - the party line is that it 'conveys DNA to RecA for recombination'. I doubt the 'for recombination' part, but lgiven DprA's presence in all the noncompetent species it must be doing something useful fro replication or repair. But DprA mutants and ComM mutants are not repair-defective (at least not especially sensitive to UV).
Maybe it's time for me to let go of my idea that both these proteins protect DNA from nucleases, and to instead start thinking more clearly about what the experimental evidence says they do. It's certainly time to reread that E. coli DprA paper (Smeets et al. 2006).
*I suspect the larger fragment is the chromosomal insert, taken up faster because it has USS.
So here's a plan: First we write out an outline of the paper we hope to publish about these results. What will this paper say? First, it will summarize the evidence that DprA and ComM protect DNA from degradation. For DprA this has been done thoroughly for Streptococcus pneumoniae; in other bacteria the dprA knockout phenotype is consistent with this but I don't know if DNA degradation has been directly measured. But the comM gene isn't present in other competent bacteria, so I think we might first need to show that it does indeed protect incoming DNA against nuclease attack.
Hmmm, I just looked at the 1998 paper describing the discovery of comM and characterization of the mutant phenotype (Gwinn et al. 1998. J. Bacteriol. 180:746-748). They did translocation assays (figure below) where cells were given linearized end-labeled DNA - one fragment cloned from the chromosome*, one vector fragment. The fragments were degraded, and the radioactivity from both fragments wound up in the chromosome, but not any faster in the comM mutant than in wildtype cells. This doesn't provide any support for my assumption that ComM protects DNA from degradation! The rec2 and comF mutants are controls, blocking translocation of DNA into the cytoplasm, and the rec1 mutation blocks homologous recombination.
So, if ComM (maybe) doesn't protect DNA from degradation, what else might it be doing that promotes recombination? And maybe DprA too - the party line is that it 'conveys DNA to RecA for recombination'. I doubt the 'for recombination' part, but lgiven DprA's presence in all the noncompetent species it must be doing something useful fro replication or repair. But DprA mutants and ComM mutants are not repair-defective (at least not especially sensitive to UV).
Maybe it's time for me to let go of my idea that both these proteins protect DNA from nucleases, and to instead start thinking more clearly about what the experimental evidence says they do. It's certainly time to reread that E. coli DprA paper (Smeets et al. 2006).
*I suspect the larger fragment is the chromosomal insert, taken up faster because it has USS.
Do bacteria become 'superbugs' in space?
Ed Yong has an article in Wired about how bacteria change gene expression when growing on the space shuttle (Space: Medicine's final frontier?). It's a well-written article but a bit credulous about the science; I don't think the data come close to justifying the interpretation.
The data were published several years ago in PNAS (Wilson et al. 2007 Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq. PNAS 104:16299-16304). The paper reports differences in gene expression when Salmonella typhimurium cells growing on the space shuttle were compared to the same bacteria growing under identical conditions on the ground. I remember thinking this was a weird result at the time, but I didn't take the trouble to carefully investigate exactly what had been done. But now I have.
The problems:
The experiments: About 7x10^6 cells of a strain of S. typhimurium that readily kills mice were pre-packed into vessels with a separate chamber containing culture medium (rich broth). In space (and simultaneously on the ground), the cells and medium were mixed and let grow and divide for 25 hr (temperature not specified but the shuttle maintains 18-27°C). The amount of cell growth is not reported but was probably about 1000-fold (assuming doubling time of ~1 hr and maximum density of ~10^10 cells/ml). In some vessels the cells were then mixed with a fixative for later microscopy and RNA analysis; in others they were mixed with enough medium to allow one more cell doubling.
Once back on earth, the fixed bacteria were examined microscopically and their RNA and proteins were extracted and amounts of specific gene transcripts and proteins measured. Mice were infected with different doses of the not-killed bacteria from both space and ground cultures, and their survival monitored.
II. The differences were due to suspension culture, not microgravity. The paper and associated media coverage, and Ed's article, describe the experiment as testing the effects of spaceflight on bacterial growth and virulence, but as best I can tell it just compared bacteria that grew suspended in medium, more or less evenly distributed, with bacteria that were initially mixed but gradually settled to the bottom of the culture vessel, where their growth would have been limited by crowding. Although in principle the microgravity of space could have had specific effects, the effects of space shuttle growth were nicely reproduced by growth on the ground in a container that gently rotated so that cells never had time to settle but did not experience the shear forces created by typical shaken cultures (Figure S5 below). (However these 'validation' cultures were done at a different temperature (37°C), and the possible effects of this are not discussed.) The authors correctly interpret this as evidence that growth in space changes gene expression because it gently prevents cells from settling, but they ignore the implications.
III. The doses of space-grown and Earth-grown bacteria used in the virulence experiments were not well controlled. The inocula used are described simply as 10^4, 10^5...10^9, but no information is given about how cell numbers were measured. This is very important because the ground-grown cells would have settled to the bottom of their container and likely grown less than the space-grown cells, which would have remained in well-mixed suspension. Simple cell counts might have been used to determine the sizes of the inocula, but these would not control for any viability differences between the space and ground cultures. Using colony counts to retrospectively estimate innocula would have controlled for any differences in viability, but then the sizes on the innocula are unlikely to have been equivalent.
I think the investigators probably just immediately inoculated the mice with ten-fold serial dilutions of the cultures, and then, after the colony counts were obtained, rounded the inocula sizes up or down to powers of ten. This means that the numbers of viable space-grown and ground-grown cells given to the mice may differed by as much as 9-fold (e.g. ground-grown bacteria at 5.5x10^6 cells/ml and space-grown bacteria at 4.5x10^7 cells/ml would both have been rounded to 10^7 cells/ml). That could easily explain the virulence differences in Figure 1 B and C, as these are substantially less than ten-fold.
IV. The evidence for changes in biofilm-forming ability is very weak. The electron micrographs Figure 1E are claimed to show that space-grown bacteria are more aggregated and clumped due to presence of an extracellular matrix. But if anything, the ground-grown cells look more aggregated, and the chunks of 'unidentified extracellular matrix' in the space-grown cells are not associated with most of the cells.
V. The culture conditions used are not at all relevant for infections. The paper makes all sorts of claims that its results have big implications for infection control during space missions. They claim that the microgravity environment of space flight and the low-shear is like that experienced by pathogens during infection of the host. However the effects of growth in space were reproduced when the cells were grown in a gently mixed ground culture. which is totally unlike ANY natural infection condition, in space or on the ground. In infections, bacteria grow on the surface of or within tissues, or in bodily fluids such as blood (a very high-shear environment). Growth during gentle suspension in rich broth is also very different than environments likely to be experienced by free-living cells in space, where bacteria are typically either surface-associated or in aerosols.
The data were published several years ago in PNAS (Wilson et al. 2007 Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq. PNAS 104:16299-16304). The paper reports differences in gene expression when Salmonella typhimurium cells growing on the space shuttle were compared to the same bacteria growing under identical conditions on the ground. I remember thinking this was a weird result at the time, but I didn't take the trouble to carefully investigate exactly what had been done. But now I have.
The problems:
- The array results were inconsistent.
- The effects were due to suspension vs settling of the cells, not microgravity.
- The doses of space-grown and Earth-grown bacteria in the virulence experiment were not well controlled and differences could account for the apparent differences in virulence.
- The evidence for changes in biofilm-forming ability is very weak.
- The culture conditions used are not at all relevant for infections.
- The difference between changes in gene expression and genetically heritable changes were not considered.
The experiments: About 7x10^6 cells of a strain of S. typhimurium that readily kills mice were pre-packed into vessels with a separate chamber containing culture medium (rich broth). In space (and simultaneously on the ground), the cells and medium were mixed and let grow and divide for 25 hr (temperature not specified but the shuttle maintains 18-27°C). The amount of cell growth is not reported but was probably about 1000-fold (assuming doubling time of ~1 hr and maximum density of ~10^10 cells/ml). In some vessels the cells were then mixed with a fixative for later microscopy and RNA analysis; in others they were mixed with enough medium to allow one more cell doubling.
Once back on earth, the fixed bacteria were examined microscopically and their RNA and proteins were extracted and amounts of specific gene transcripts and proteins measured. Mice were infected with different doses of the not-killed bacteria from both space and ground cultures, and their survival monitored.
The microarray analysis of gene expression looks methodologically OK: Because Salmonella has about 4300 genes, microarray experiments of gene expression need to be carefully controlled and replicated to eliminate false-positive differences in gene expression. The authors did three biological replicates (three different cultures from space and from ground.
I. The array results were inconsistent. Some Hfq-regulated genes were found to be up-regulated in suspension (space) culture, and some were down-regulated. This isn't itself a problem, as Hfq is known to have different effects on different genes. But Table 1 doesn't tell us which genes are expected to be up-regulated and which down-regulated, so we can't easily compare the prediction with the result. I picked a few genes at random and compared the effect of space growth to the effect of a hfq deletion in E. coli (Guisbert et al 2007). Most of the genes listed as Hfq-regulated in Table 1 weren't listed in this paper. Of those that were, most had effects in the same direction, but some were in opposite directions (e.g. adhE was up 4.75-fold in space, but down 2.8-fold in a hfq deletion).
II. The differences were due to suspension culture, not microgravity. The paper and associated media coverage, and Ed's article, describe the experiment as testing the effects of spaceflight on bacterial growth and virulence, but as best I can tell it just compared bacteria that grew suspended in medium, more or less evenly distributed, with bacteria that were initially mixed but gradually settled to the bottom of the culture vessel, where their growth would have been limited by crowding. Although in principle the microgravity of space could have had specific effects, the effects of space shuttle growth were nicely reproduced by growth on the ground in a container that gently rotated so that cells never had time to settle but did not experience the shear forces created by typical shaken cultures (Figure S5 below). (However these 'validation' cultures were done at a different temperature (37°C), and the possible effects of this are not discussed.) The authors correctly interpret this as evidence that growth in space changes gene expression because it gently prevents cells from settling, but they ignore the implications.
III. The doses of space-grown and Earth-grown bacteria used in the virulence experiments were not well controlled. The inocula used are described simply as 10^4, 10^5...10^9, but no information is given about how cell numbers were measured. This is very important because the ground-grown cells would have settled to the bottom of their container and likely grown less than the space-grown cells, which would have remained in well-mixed suspension. Simple cell counts might have been used to determine the sizes of the inocula, but these would not control for any viability differences between the space and ground cultures. Using colony counts to retrospectively estimate innocula would have controlled for any differences in viability, but then the sizes on the innocula are unlikely to have been equivalent.
I think the investigators probably just immediately inoculated the mice with ten-fold serial dilutions of the cultures, and then, after the colony counts were obtained, rounded the inocula sizes up or down to powers of ten. This means that the numbers of viable space-grown and ground-grown cells given to the mice may differed by as much as 9-fold (e.g. ground-grown bacteria at 5.5x10^6 cells/ml and space-grown bacteria at 4.5x10^7 cells/ml would both have been rounded to 10^7 cells/ml). That could easily explain the virulence differences in Figure 1 B and C, as these are substantially less than ten-fold.
IV. The evidence for changes in biofilm-forming ability is very weak. The electron micrographs Figure 1E are claimed to show that space-grown bacteria are more aggregated and clumped due to presence of an extracellular matrix. But if anything, the ground-grown cells look more aggregated, and the chunks of 'unidentified extracellular matrix' in the space-grown cells are not associated with most of the cells.
V. The culture conditions used are not at all relevant for infections. The paper makes all sorts of claims that its results have big implications for infection control during space missions. They claim that the microgravity environment of space flight and the low-shear is like that experienced by pathogens during infection of the host. However the effects of growth in space were reproduced when the cells were grown in a gently mixed ground culture. which is totally unlike ANY natural infection condition, in space or on the ground. In infections, bacteria grow on the surface of or within tissues, or in bodily fluids such as blood (a very high-shear environment). Growth during gentle suspension in rich broth is also very different than environments likely to be experienced by free-living cells in space, where bacteria are typically either surface-associated or in aerosols.
VI. The differences between changes in gene expression and genetically heritable (mutational) change were not considered at all. This is a major oversight, confounding physiological and evolutionary changes. If the space-grown bacteria are just in an altered physiological state, they might be better able to initiate an infection but they would quickly alter their physiology to that determined by the host environment. If the differences were due to mutations favoured during growth in space, then we might have to worry about 'superbugs'. But the cells would have gone through only about 10 doublings, so it's very unlikely that any new mutations had accumulated to significant levels.
Overall I'm not convinced that this paper has any implications for space medicine at all. It certainly doesn't show that growth in space transforms bacteria into superbugs.
How to visit the Creation Museum and cost them money
While I was in Louisville last week my host at the lovely Columbine Inn told me about his reluctant visit to the nearby Creation Museum. He was dragged there by a friend, on condition that she pay his admission so he wouldn't be giving them any financial support.
BUT, he discovered an online 2-for-1 admission coupon. So he printed out a whole stack of them and handed them out in the parking lot!
Unfortunately the museum staff must have caught on, because I can't find the coupon anywhere on their site.
BUT, he discovered an online 2-for-1 admission coupon. So he printed out a whole stack of them and handed them out in the parking lot!
Unfortunately the museum staff must have caught on, because I can't find the coupon anywhere on their site.
State of the Research II: The post-doc's projects
Not surprisingly, the post-doc has a more ambitious collection of projects than the new grad student does. He's on the other side of the continent visiting our collaborator and learning how to set up a whatchamacallit (an assembly line for analyzing data...). I'll see if I can summarize all his projects.
His first project is the analysis of recombination tracts. This relies on full-genome sequencing of individual recombinants between our standard lab strain Rd (the recipient) and a clinical H. influenzae isolate that differs at about 2% of alignable positions and by several thousand indels and other structural variations. The first paper, describing analysis of the first four recombinants, is ready to be submitted (tomorrow?), and the multiplexed sequencing of another 60 or so strains should be producing its first results this week (done by the Genome Sciences Centre under our Genome BC grant). (There was a concern about the quality of the libraries, so one lane (8 genomes) is being done ahead of the others to check this.)
His second project is the analysis of recombination frequencies across the genome, for every position that differs between the same two strains. We originally planned to include this with the Genome BC work, but UBC's Biodiversity Centre has a new sequencer and is offering longer cheaper sequencing runs that would be very well suited for this project. BUT, to use their service we'd have to make our own libraries (i.e. the post-doc would have to make his own libraries, or persuade the collaborator's technician to make them for him). It might be better to just do the first pass with the Genome BC/Genome Sciences Centre as planned. But I think the experiments to prepare the DNA that will be sequenced haven't even been started yet. In principle these should be straightforward extensions of the work he's already optimized, but I'm not counting on that.
His third project is the 'QTL-mapping' project, using identification of donor sequences in recombinants between the two strains to map the loci responsible for phenotypic differences. The first phenotypic difference he's looking at is the >1000-fold difference in transformability between the two strains; the second will be (we hope) sensitivity to killing by serum. He's doing this with help from an undergrad (almost-grad), who has been developing the screening method that will identify recombinant strains with reduced transformation. Then now have an assay that should work and that is only moderately more troublesome than what we originally planned. They're going to screen the same strains that are being sequenced for the first project, so here's hoping they find some transformation changes.
His fourth project is the analysis of uptake specificity. The first step of this was very successful, and he's subsequently put a LOT of work into developing methods to analyze the data. We won't do any more sequencing on this project until we get our new DNA uptake grant (or until the reviewers reject it but advise doing additional work). I'm eager to get the present results written up and published asap, but he keeps finding new analyses that he thinks need to be done.
His first project is the analysis of recombination tracts. This relies on full-genome sequencing of individual recombinants between our standard lab strain Rd (the recipient) and a clinical H. influenzae isolate that differs at about 2% of alignable positions and by several thousand indels and other structural variations. The first paper, describing analysis of the first four recombinants, is ready to be submitted (tomorrow?), and the multiplexed sequencing of another 60 or so strains should be producing its first results this week (done by the Genome Sciences Centre under our Genome BC grant). (There was a concern about the quality of the libraries, so one lane (8 genomes) is being done ahead of the others to check this.)
His second project is the analysis of recombination frequencies across the genome, for every position that differs between the same two strains. We originally planned to include this with the Genome BC work, but UBC's Biodiversity Centre has a new sequencer and is offering longer cheaper sequencing runs that would be very well suited for this project. BUT, to use their service we'd have to make our own libraries (i.e. the post-doc would have to make his own libraries, or persuade the collaborator's technician to make them for him). It might be better to just do the first pass with the Genome BC/Genome Sciences Centre as planned. But I think the experiments to prepare the DNA that will be sequenced haven't even been started yet. In principle these should be straightforward extensions of the work he's already optimized, but I'm not counting on that.
His third project is the 'QTL-mapping' project, using identification of donor sequences in recombinants between the two strains to map the loci responsible for phenotypic differences. The first phenotypic difference he's looking at is the >1000-fold difference in transformability between the two strains; the second will be (we hope) sensitivity to killing by serum. He's doing this with help from an undergrad (almost-grad), who has been developing the screening method that will identify recombinant strains with reduced transformation. Then now have an assay that should work and that is only moderately more troublesome than what we originally planned. They're going to screen the same strains that are being sequenced for the first project, so here's hoping they find some transformation changes.
His fourth project is the analysis of uptake specificity. The first step of this was very successful, and he's subsequently put a LOT of work into developing methods to analyze the data. We won't do any more sequencing on this project until we get our new DNA uptake grant (or until the reviewers reject it but advise doing additional work). I'm eager to get the present results written up and published asap, but he keeps finding new analyses that he thinks need to be done.
State of the research: I. the new grad student's project
Before I get back into doing experiments I need to reestablish my knowledge of what everyone else in the lab is working on. So I'm going to do a quick summary series of posts, starting with the new grad student's warm-up project. (This isn't his real thesis project, but a smaller project he took on as a way to get a better grip on the lab's goals and techniques.)
The big-picture goal of the project is to find out how the cytoplasmic proteins DprA and ComM protect incoming DNA. Both genes are in H. influenzae's CRP-S competence regulon, and knockouts have similar phenotypes -- DNA uptake and translocation are normal, but the transformation frequency is severely reduced. I initially hypothesized that DprA acts by directly blocking a specific cytoplasmic nuclease that would otherwise degrade free DNA strands in the cytoplasm, but work in S. pneumoniae has indicated that it instead binds to DNA, shielding it from nucleases. The DprA protein is ubiquitous in Bacteria (competent and not) and in the well-studied competent model systems knockouts consistently give an uptake+ transformation- phenotype. The E.coli protein is reported to partly restore competence to an H. influenzae knockout(Smeets et al 2006). This was interpreted as meaning that competent cells are co-opting a DprA function independent of DNA uptake, but I have two concerns with this. First, the wildtype transformation frequency in this experiment was about 500-fold lower than it should be. Second, E. coli has a competence regulon (though dprA isn't in it), so its DprA may not be the best example of a competence-independent protein.
We don't know anything about what ComM does (it's not involved in other competence systems, and homologs aren't known from other systems), but our hypothesis is that, like DprA, it protects DNA from degradation by cytoplasmic nucleases, either by sequestering the DNA or blocking/inactivating one or more nucleases. ComM is a member of a widely distributed family of proteins with many different functions (the AAA+-superfamily); here's the first two sentences from a recent review by Snider et al.:
I began the experiments about seven years ago by testing the ExoV nuclease encoded by the recB, recC and recD genes, because this nuclease is known to be blocked by specific phage-encoded proteins (it otherwise prevents phage reproduction by degrading phage DNA). The results were negative, and an undergraduate (Stephanie, I think) then repeated the tests with ComM, again with negative results. This was more than 5 years ago. A few years ago, a different undergraduate worked on extending the results to several other candidate nucleases for which knockouts had become available (Kumar et al 2007), and now the new grad student is finishing that work.
Four different exonucleases could in principle contribute to cytoplasmic DNA degradation: ExoV, ExoI, ExoVII and RecJ. The role of ExoVII will be tricky to investigate because its H. influenzae knockout was found to be lethal, so the grad student's goal is to compare the transformation phenotypes of dprA-knockout and comM-knockout cells with and without knockouts of exoI, recJ and exoI+recJ.
His first task was to locate or construct the various strains he needed. By the beginning of March he had made and PCR-verified all the strains. He was originally going to also test the exonuclease mutations in a dprA/comM double mutant, but ran into problems measuring its transformation frequency without the nuclease mutations (too low). Of course he could still check whether the transformation frequency became much higher when one or more nuclease is knocked out, but that's relatively low priority, at least until the dprA-nuclease and comM-nuclease combinations have been tested separately.
I'll find out tomorrow how far things have progressed....
The big-picture goal of the project is to find out how the cytoplasmic proteins DprA and ComM protect incoming DNA. Both genes are in H. influenzae's CRP-S competence regulon, and knockouts have similar phenotypes -- DNA uptake and translocation are normal, but the transformation frequency is severely reduced. I initially hypothesized that DprA acts by directly blocking a specific cytoplasmic nuclease that would otherwise degrade free DNA strands in the cytoplasm, but work in S. pneumoniae has indicated that it instead binds to DNA, shielding it from nucleases. The DprA protein is ubiquitous in Bacteria (competent and not) and in the well-studied competent model systems knockouts consistently give an uptake+ transformation- phenotype. The E.coli protein is reported to partly restore competence to an H. influenzae knockout(Smeets et al 2006). This was interpreted as meaning that competent cells are co-opting a DprA function independent of DNA uptake, but I have two concerns with this. First, the wildtype transformation frequency in this experiment was about 500-fold lower than it should be. Second, E. coli has a competence regulon (though dprA isn't in it), so its DprA may not be the best example of a competence-independent protein.
We don't know anything about what ComM does (it's not involved in other competence systems, and homologs aren't known from other systems), but our hypothesis is that, like DprA, it protects DNA from degradation by cytoplasmic nucleases, either by sequestering the DNA or blocking/inactivating one or more nucleases. ComM is a member of a widely distributed family of proteins with many different functions (the AAA+-superfamily); here's the first two sentences from a recent review by Snider et al.:
The AAA+ proteins (‘ATPases associated with diverse cellular activities’) form a large and diverse superfamily found in all organisms. These proteins typically assemble into hexameric ring complexes that are involved in the energy-dependent remodeling of macromolecules.The series of experiments we've been doing asks which nucleases DprA and ComM protect DNA from. The basic experiment is simple genetic test: find out whether knocking out each candidate nuclease increases transformation in a dprA or comM knockout background. If transformation is higher when the nuclease is inactive, and the effect is much stronger when DprA or ComM is also missing, then DprA or ComM normally protect DNA from the nuclease.
I began the experiments about seven years ago by testing the ExoV nuclease encoded by the recB, recC and recD genes, because this nuclease is known to be blocked by specific phage-encoded proteins (it otherwise prevents phage reproduction by degrading phage DNA). The results were negative, and an undergraduate (Stephanie, I think) then repeated the tests with ComM, again with negative results. This was more than 5 years ago. A few years ago, a different undergraduate worked on extending the results to several other candidate nucleases for which knockouts had become available (Kumar et al 2007), and now the new grad student is finishing that work.
Four different exonucleases could in principle contribute to cytoplasmic DNA degradation: ExoV, ExoI, ExoVII and RecJ. The role of ExoVII will be tricky to investigate because its H. influenzae knockout was found to be lethal, so the grad student's goal is to compare the transformation phenotypes of dprA-knockout and comM-knockout cells with and without knockouts of exoI, recJ and exoI+recJ.
His first task was to locate or construct the various strains he needed. By the beginning of March he had made and PCR-verified all the strains. He was originally going to also test the exonuclease mutations in a dprA/comM double mutant, but ran into problems measuring its transformation frequency without the nuclease mutations (too low). Of course he could still check whether the transformation frequency became much higher when one or more nuclease is knocked out, but that's relatively low priority, at least until the dprA-nuclease and comM-nuclease combinations have been tested separately.
I'll find out tomorrow how far things have progressed....
TEDxWaterloo on Open Science: Michael Nielsen
http://www.youtube.com/watch?v=DnWocYKqvhw
Initially talking about a 'Polymath' project, successful
Then about a quantum wiki, proposed at a conference. People liked the idea but hoped that others would do the work, make the contributions. So it went nowhere. Social networks for scientists usually fail too.
Why? Science is competitive, to get a job you gain much more by writing a paper (even a lousy one) than by contributing to a public project. The end product of the polymath project was conventional papers.
1990s, a successful collaborative genetics project. How did it overcome the reluctance to upload data for shared use. The Bermuda principles: human genetics data should be uploaded and publicly available. Solidified in policy, by NIH and Wellcome, to work on project had to agree to these principles.
But much genomic data is hoarded, also computer programs, description of projects. Open Science movement wants to change this. How to change science?
Galileo example: Kept discovery secret, but distributed in code (anagram) to ensure claim of priority. Later 18th and 19th century, struggle to make science public. (I think he's saying this was an open science revolution...)
Now have new tools. What can you do to help start the second open science revolution? Use the new tools. But be very generous in giving credit to others for sharing. Promote by conversations to change the culture of science.
Single most important thing we can do is raise public awareness of importance/value of openness. Talk it up all around you.
('Consciousness raising' - did it work for feminism?)
Initially talking about a 'Polymath' project, successful
Then about a quantum wiki, proposed at a conference. People liked the idea but hoped that others would do the work, make the contributions. So it went nowhere. Social networks for scientists usually fail too.
Why? Science is competitive, to get a job you gain much more by writing a paper (even a lousy one) than by contributing to a public project. The end product of the polymath project was conventional papers.
1990s, a successful collaborative genetics project. How did it overcome the reluctance to upload data for shared use. The Bermuda principles: human genetics data should be uploaded and publicly available. Solidified in policy, by NIH and Wellcome, to work on project had to agree to these principles.
But much genomic data is hoarded, also computer programs, description of projects. Open Science movement wants to change this. How to change science?
Galileo example: Kept discovery secret, but distributed in code (anagram) to ensure claim of priority. Later 18th and 19th century, struggle to make science public. (I think he's saying this was an open science revolution...)
Now have new tools. What can you do to help start the second open science revolution? Use the new tools. But be very generous in giving credit to others for sharing. Promote by conversations to change the culture of science.
Single most important thing we can do is raise public awareness of importance/value of openness. Talk it up all around you.
('Consciousness raising' - did it work for feminism?)
The American Libraries Association pegs my irony meter
The American Libraries Association has just published what looks like an excellent report on Open Access publishing: Open access: what you need to know now, by Walt Crawford. It's only 80 pages, much of which is available on Google Books. (Thanks to Metadata for posting about this.)
The irony? The ALA could easily provide this report as a pdf for free, but instead they're selling it for $45! For a 80-page paperback! There's also an e-book version, $36!)
The irony? The ALA could easily provide this report as a pdf for free, but instead they're selling it for $45! For a 80-page paperback! There's also an e-book version, $36!)
I've just looked at the author's blog, Walt at Random. I was hoping to find some expression of concern about this pricing but, although he's an engaging writer with interesting ideas, he seems more concerned with book sales than with readership.
Preparing my talk about scientific communication
I'm revising/improving my plans for the first talk I'm preparing: 'What I learned from #arseniclife: communication and quality control in science'.
I still plan to start by going over the Wolfe-Simon debacle, from NASA's first press release to the present state of affairs (10-15 minutes). But I've reorganized my walk-through of all the ways science is communicated (to scientists and to the public) and the ways quality is maintained. Rather than using a semi-historical framework overall, it's now a series of overlapping issues: Publication, Access, Sharing, Searching, Publicity, Pre-publication review, Post-publication review, and six more. Each will be shown as a one-slide mini-history. But the histories are not so much of changes (we used to do that, but now we do this) as of broadening options (we used to only do that, but now we can also do this and this and this). This should probably be given no more than about 15 minutes, so I'll need to trim down my list of issues.
Then I'll list the roles these issues played in #arseniclife, first slow (funding, collaborative research, manuscript, peer review, acceptance) then fast (press release, press conference, in-press publication, excited articles in the media, critical peer review on blogs, spread by Twitter, critical articles in the media, access first by paywall then open) then slow (journal articles..., formal publication stalled).
Then the positive and negative effects. Positive: Communication between scientists was very efficient, and experts very quickly reached a strong consensus that the the conclusion was wrong. The media coverage provided a very public demonstration of how science is self-correcting. Negative: Many members of the public took this as a demonstration of how science gets things wrong, and many more completely missed the correction, seeing only the original story.
But this isn't a very good model for 'normal science'. Rather it's a warning of what can go wrong if you reach too high. Most papers never attract comments on the journal sites (none of the ten year-old PLoS ONE papers I checked had any). Substantial discussion happens on blogs; the Research Blogging aggregator site linked to discussions of 31 articles on March 31 and 35 on March 23. The posts are excellent, but most of them have few or no comments, and I don't know how often they are even seen by the authors of the articles.
I'd like to consider the good and bad outcomes of online publication, though I may not have time. One of these is the problems with for-profit journals. Yesterday I let myself get distracted by the 'Frontiers in ...' enterprise, tabulating data on how many papers each 'journal' has published and how many people are on its Editorial Board. With the exception of the original Frontiers in Neuroscience, the ratio is at least 10 board members for each paper published, so this appears to be mainly a resource for resume-padding, not scientific communication. Most of the Bentham group journals are just as bad, averaging one article per year with an Editorial Board of about 100.
I want to end with considering how human nature limits our use of the new forms of communication to promote the advance of scientific knowledge, and how individuals and institutions can intervene. One way is by having publishers and granting agencies enforce community standards (data deposition, open-access publishing). Research blogging certainly won't hurt, but we need to find ways to bring it into the mainstream.
I still plan to start by going over the Wolfe-Simon debacle, from NASA's first press release to the present state of affairs (10-15 minutes). But I've reorganized my walk-through of all the ways science is communicated (to scientists and to the public) and the ways quality is maintained. Rather than using a semi-historical framework overall, it's now a series of overlapping issues: Publication, Access, Sharing, Searching, Publicity, Pre-publication review, Post-publication review, and six more. Each will be shown as a one-slide mini-history. But the histories are not so much of changes (we used to do that, but now we do this) as of broadening options (we used to only do that, but now we can also do this and this and this). This should probably be given no more than about 15 minutes, so I'll need to trim down my list of issues.
Then I'll list the roles these issues played in #arseniclife, first slow (funding, collaborative research, manuscript, peer review, acceptance) then fast (press release, press conference, in-press publication, excited articles in the media, critical peer review on blogs, spread by Twitter, critical articles in the media, access first by paywall then open) then slow (journal articles..., formal publication stalled).
Then the positive and negative effects. Positive: Communication between scientists was very efficient, and experts very quickly reached a strong consensus that the the conclusion was wrong. The media coverage provided a very public demonstration of how science is self-correcting. Negative: Many members of the public took this as a demonstration of how science gets things wrong, and many more completely missed the correction, seeing only the original story.
But this isn't a very good model for 'normal science'. Rather it's a warning of what can go wrong if you reach too high. Most papers never attract comments on the journal sites (none of the ten year-old PLoS ONE papers I checked had any). Substantial discussion happens on blogs; the Research Blogging aggregator site linked to discussions of 31 articles on March 31 and 35 on March 23. The posts are excellent, but most of them have few or no comments, and I don't know how often they are even seen by the authors of the articles.
I'd like to consider the good and bad outcomes of online publication, though I may not have time. One of these is the problems with for-profit journals. Yesterday I let myself get distracted by the 'Frontiers in ...' enterprise, tabulating data on how many papers each 'journal' has published and how many people are on its Editorial Board. With the exception of the original Frontiers in Neuroscience, the ratio is at least 10 board members for each paper published, so this appears to be mainly a resource for resume-padding, not scientific communication. Most of the Bentham group journals are just as bad, averaging one article per year with an Editorial Board of about 100.
I want to end with considering how human nature limits our use of the new forms of communication to promote the advance of scientific knowledge, and how individuals and institutions can intervene. One way is by having publishers and granting agencies enforce community standards (data deposition, open-access publishing). Research blogging certainly won't hurt, but we need to find ways to bring it into the mainstream.
Two talks
The first talk I'm preparing has the title 'What I learned from #arseniclife: communication and quality control in science'. My current plan is to spend 10-15 minutes going over the Wolfe-Simon debacle, from NASA's first press release to the present state of affairs (paper still not published).
Then I'll do a semi-historical walk-through of all the ways science is communicated (to scientists and to the public) and the ways quality is maintained. I'll organize it historically, but I think every method can be illustrated with a present-day example, because although we post electronic critiques we still have print-only journals (though I'll probably have to ask a librarian for examples of these). As I go through these I'll illustrate as many as I can from the #arseniclife story. This should be given about 20 minutes.
Then I'll talk about how the new options have good and bad effects on the advance of scientific knowledge, and how optimizing these is limited by human nature. And maybe end with some ideas of how we, as individual scientists, can make the most of these.
The second talk has my usual provocative title 'Do bacteria have sex?' In the past this talk has been mainly about the regulation of competence, but now I want to extend it to include our newer work on recombination tracts and on uptake specificity. So I'm going to shorten the introduction a bit, about why whether bacteria have sex is important and why natural competence is the only parasexual process needing investigation. And I'll shorten the regulation section too. Then I'll talk about how we still need to understand the recombinational consequences of parasexual processes, because these are very significant for bacterial populations and evolution, and show our new recombination-tract data.
Then I'll bring up the uptake-specificity issue (maybe I'll foreshadow this earlier), as the one remaining problem with the evidence that bacteria take up DNA as food. I'll emphasize that it only applies to two small groups of bacteria, but is still considered by many as a compelling reason to assume that all bacteria take up DNA for recombination. Maybe I'll go back to the candyland movie of the uptake mechanism (make a short version, with just the uptake step). I'll propose the model that uptake specificity is a mechanistic solution to a physical problem, and then describe the new analysis.
As described above, this has the potential to be much too long. But I should aim for 45 minutes, to give lots of time for questions.
Then I'll do a semi-historical walk-through of all the ways science is communicated (to scientists and to the public) and the ways quality is maintained. I'll organize it historically, but I think every method can be illustrated with a present-day example, because although we post electronic critiques we still have print-only journals (though I'll probably have to ask a librarian for examples of these). As I go through these I'll illustrate as many as I can from the #arseniclife story. This should be given about 20 minutes.
Then I'll talk about how the new options have good and bad effects on the advance of scientific knowledge, and how optimizing these is limited by human nature. And maybe end with some ideas of how we, as individual scientists, can make the most of these.
The second talk has my usual provocative title 'Do bacteria have sex?' In the past this talk has been mainly about the regulation of competence, but now I want to extend it to include our newer work on recombination tracts and on uptake specificity. So I'm going to shorten the introduction a bit, about why whether bacteria have sex is important and why natural competence is the only parasexual process needing investigation. And I'll shorten the regulation section too. Then I'll talk about how we still need to understand the recombinational consequences of parasexual processes, because these are very significant for bacterial populations and evolution, and show our new recombination-tract data.
Then I'll bring up the uptake-specificity issue (maybe I'll foreshadow this earlier), as the one remaining problem with the evidence that bacteria take up DNA as food. I'll emphasize that it only applies to two small groups of bacteria, but is still considered by many as a compelling reason to assume that all bacteria take up DNA for recombination. Maybe I'll go back to the candyland movie of the uptake mechanism (make a short version, with just the uptake step). I'll propose the model that uptake specificity is a mechanistic solution to a physical problem, and then describe the new analysis.
As described above, this has the potential to be much too long. But I should aim for 45 minutes, to give lots of time for questions.
Classes are over!
Yesterday was the last lecture for my pilot genetics course, so now I can turn my attention (some of it, there's still the final) to research-related stuff.
Next week I'm giving two talks at the University of Louisville (Kentucky). One will be on competence (Do bacteria have sex?); I'll show my stop-motion animation of DNA uptake and work in the new results on recombination tracts and uptake specificity. The other is titled 'What I learned from #arseniclife: communication and quality control in science'. I've not formally talked about this before, so I still have to sort out what I did learn.
The post-doc's paper on recombination tracts has been provisionally accepted by PLoS Pathogens, and he's working on the revisions. One highlight was a reviewer who wrote "I wished for death" but nevertheless recognized the value of our meticulously detailed description of the control analyses.
Maybe if I go stand in front of my bench I'll be able to remember what I was working on before classes started...
Next week I'm giving two talks at the University of Louisville (Kentucky). One will be on competence (Do bacteria have sex?); I'll show my stop-motion animation of DNA uptake and work in the new results on recombination tracts and uptake specificity. The other is titled 'What I learned from #arseniclife: communication and quality control in science'. I've not formally talked about this before, so I still have to sort out what I did learn.
The post-doc's paper on recombination tracts has been provisionally accepted by PLoS Pathogens, and he's working on the revisions. One highlight was a reviewer who wrote "I wished for death" but nevertheless recognized the value of our meticulously detailed description of the control analyses.
Maybe if I go stand in front of my bench I'll be able to remember what I was working on before classes started...
Is Felisa Wolfe-Simon an Alien?
An anonymous source just sent me this very interesting document (they can be contacted at felisa.walks.among.us@gmail.com):
“A Bacterium That Can Grow by Using Arsenic Instead of Phosphorous”“This paper is silly” (Grace, 2011). We think Felisa is an alien (Fig. 1,pg 5) and this is why she has written such a flawed paper; because she is trying to hide the existence of her type by making scientists pick holes in her experimental design. Consequently these scientists will not believe her experiment , and therefore the existence of aliens will not be believed as there is only flawed evidence in favour of this possibility.This is why the bacteria strain is called GFAJ-1; “Give Felisa a Job”, because Felisa is not human and would like a job in order to disguise her true identity and to be accepted in to the human race, like Clark Kent (Fig. 2, page 5). Additionally this job will enable her to gain the trust of humans in order to gain a better understanding of our planet.This paper is clearly designed to distract NASA and other microbiologists from the search for alien communications on to simple arsenate-based life. This is to allow the complex alien species to invade earth. Furthermore, the lack of simple controls and correct washing techniques is to create conflict in the scientific community to further distract scientists from the reality of extra-terrestrial life. Of course, it is also quite possible that NASA funded this paper because they know of the impending alien invasion but want a place in the New World Order.
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