RRResearch

CIHR proposal - mutant phenotypes

2012-02-13T09:00:00.000-08:00

We've been going back and forth and around and around on the part of our CIHR grant proposal where we propose to ... well, part of the problem is that we've not decided whether this section should just propose to do one unified set of analyses or add in various disparate analyses that don't fit elsewhere.

There is a unified set of analyses to be done. We have a complete set of knockout mutants that have been only partially characterized (transformation assays, preliminary DNA uptake assays). So we're proposing to do a more thorough analysis of all the mutants whose DNA uptake is defective (because DNA uptake is the overall focus of the proposal), using two new methods.

One problem of working with the standard lab strain of H. influenzae is that it doesn't make long pili that can be seen by electron microscopy (EM), which means we can't tell whether our mutants block the assembly of pilin subunits into the pseudopili that pull DNA into the cell. (Well, we might be able to devise an assay for pseudopili, using crosslinking, but this will be a fallback.) So we're going to use transformation to put each of our knockout mutations into the NP strain, which does make pili, and then use EM to see if the mutation prevents pilus assembly. We'll be especially interested in mutants with abnormal pili.
Our DNA uptake assays use centrifugation (pellet cells, resuspend pellet in fresh liquid, repeat twice) to wash unbound DNA away from cells, with or without first adding DNase I to digest DNA that's not been taken into the cell. But a B. subtilis paper I read on Saturday described instead washing cells by filtration, using special 96-well plates that have a filter in each well. This allows more thorough washing and is gentler on the cells. We need to repeat and replicate the uptake assays on our uptake-defective mutants anyway, because we've decided these are critical to detecting whether the mutants can still bind DNA at the cell surface. So we're now proposing to use the filtration assay to get very solid data for all these mutants.

These assays will let us distinguish proteins that are needed for pilus assembly from proteins that matter only after the pilus has been assembled. We can describe what we expect to find, based on postulated protein homologies and our work so far, and explain how the results will be interpreted. So far so good.

The hard part is deciding what other investigations to include in this section. One issue raised by a reviewer of the previous submission is that knockout mutants are a very crude tool for investigating function, especially for processes that depend on concerted work by many components. In competence, a knockout that eliminates the pre-pilin peptidase has the same uptake and transformation phenotype as one that eliminates the pilin subunits or the assembly ATPase or the outer membrane pore used by the pseudopilus. We've added another phenotype to our screen (production of pili by strain NP), but we would like to have at least one experiment showing that we can use less-drastic mutations to investigate the specific function of a gene.

I think we should describe several analyses we know we want to do (each a short paragraph), and explain that similar techniques can be applied to other genes, depending on the phenotypes revealed by assays 1 and 2 above. I was planning to organize these by the questions they address, but it might be more effective to organize them by the techniques they illustrate.

Different DNA substrates for the uptake assays: DNA concentration, ±USS, short vs long DNA fragments to detect retraction and retention defects (e.g. for the comE1 mutant), end blocked with a bead to detect polarity...
Double mutants: To see if the DNA uptake by the comE1 knockout depends on DNA translocation across the inner membrane we'll test a comE1-rec2 double mutant.
Reisolation of DNA from the periplasm: Test whether comF is blocked at the same step as rec2.
Truncation mutations:
Point mutations that change specific amino acids: We can make specific mutations to change proposed DNA-binding residues in the secretin subunits that form the outer membrane pore (comE). We can test whether pilF2 encodes a pilotin homolog by mutating the specific residue that should be its lipidation site.
Random mutagenesis of a gene or part of a gene, followed by screening for a desired phenotype: We'd mutagenize the gene in an expression plasmid, and then put the plasmid into the corresponding knockout mutant to look for the desired phenotype. We're considering doing this to pilB to screen for an effect on retraction, but this is a long shot.
Cross-species complementation and optical tweezer experiments will be described in separate sections, but they might be mentioned here.

OK, I think writing this post has given me a workable plan.

Pinterest report (ho hum)

2012-02-12T10:12:00.000-08:00

Thanks to an invitation from @SciChem_, I got a Pinterest account yesterday and tried it out.

I wasn't hoping that Pinterest would be a good substitute for formal reference-management programs like Mendeley or Endnote. Instead I was looking for a way to remind myself about research papers that might be important or useful for specific projects - an electronic improvement on printing out pdfs and spreading them all over the floor of my office. Bottom line: it's not very flexible but still might be useful.

It's very easy to use. You open an account (you can ask me for an invitation), create one or more blank 'boards', and drag a 'Pin it' button to the bookmarks bar of your browser. Then, whenever you see an online image you'd like to remember, you click the button. This brings up a little a Pinterest window that lets you identify which of the images on the page you want to put on your board, and what text you want to appear below the image. Here's the Pinterest board I created for recent papers about the molecular motors associated with Type 4 pili.

One difficulty is that Pinterest only recognizes images on web pages. For most papers that's not a problem, you just open the html view, click the button and decide which figure you want on your board. But this strategy didn't work for PLoS papers (at least not PLoS Biology), since their html files don't contain anything that Pinterest can recognize as a suitable image. Instead their figures are represented by thumbnails linked to large figures that Pinterest doesn't see. I couldn't figure out any straightforward way to pin images from these html files.

A more general problem is Pinterest's inflexibility (the down side of its simplicity). Users have very little control over anything except deciding what boards to have and what images to pin on them. There's no way to control image size and placement, or board appearance. Pinterest also pushes its social agenda annoyingly hard - boxes appear demanding that you assign your boards to categories so others can find them, and they won't go away until you do.You're pushed to 'follow' other users and to comment on whatever people put on their boards.

When would Pinterest be useful for a scientist? Anytime you're searching the web for resources, it lets you keep an easy-to-share visual log of what you've found. My display of recent type 4 pili papers helps me remember what to read when I write that part of my grant proposal. If I was going to a conference in a far-away place, I might use it to gather ideas for recreational activities and then email the board's link to friends who might be interested in doing them with me. For group projects you can also set up a group board with several authorized contributors.

HI0569, gene of mystery

2012-02-12T06:57:00.000-08:00

The RA's heroic project to create knockout mutants of every gene in the H. influenzae competence regulon has turned up one big surprise - the HI0659 gene. This small cytoplasmic protein, whose mRNA was induced about 25-fold in competent cells in our old microarray experiments, turns out to be essential for competence. The knockout mutant doesn't detectably take up DNA and produces no transformants.

The first question to consider (and maybe to answer) is whether this protein plays an essential mechanistic role in DNA uptake or has a regulatory function that's needed for effective expression of the other genes. It's only 98 amino acids long, and most of that is a single helix-turn-helix (HTH) domain (see figure). HTH domains typically regulate gene expression by binding to specific DNA sequences, but they usually are only part of larger proteins whose activities are in turn regulated by other effectors such as sugars and amino acids. But HI0659 doesn't have much room for other interactions, decreasing the likelihood of a regulatory function. On the other hand, the protein doesn't have much room for DNA uptake functions either. And it doesn't have any targeting signals that would send it into the cell envelope.

The postdoc speculates that it might bind RNA rather than DNA, perhaps interacting with sxy mRNA. Apparently some HTH motifs do bind RNA. But ssRNA has a very different structure than dsDNA, so I wonder if what these motifs bind is actually dsRNA.

One thing the microarray summary doesn't tell us is the basal level of HI0659 mRNA expression. This is of interest because, if it's a regulator of competence that's needed for expression of the other genes in the CRP-S regulon, it should be active before they come on. Maybe it's active constitutively at a moderate level, and induced even higher in competent cells.

It's just downstream of HI0660, another tiny protein with no known function. HI0660 is even less conserved than I0659, and knockouts of it have normal competence. Surprisingly, the 'marked' knockout of HI0659 (the same deletion with an inserted SpcR cassette) retains some competence.

Here's a graphic of HI0660 and HI0659 aligned with homologs. I asked the database to find homologs in other Pasturellacean species (H. ducreyi, Mannheimia succinoproducens, Pasteurella multocida) but none were found even though I lowered the % similarity cutoff to 30% from the default 40%. This is a bit surprising, because A. pleuropneumoniae is a more distant relative than M. succinoproducens and P. multocida. The operon is also in M. haemolytica, a close relative of A. pleuropneumoniae not shown in the figure, and I think the grad student who did the analysis also found that it has a CRP-S promoter in these species.

For the CIHR proposal we're going to propose to do 'RNA-seq' of the HI0659 knockout and controls, to look for changes in the mRNA population. RNA-seq is the shorthand term for measuring the abundances of all a cell's transcripts by doing deep sequencing of a cDNA prep. It's pretty straightforward; the only big problem is avoiding wasteful sequencing of ribosomal RNAs, which are by far the most abundant RNAs in bacterial cells, but the postdoc says there's a good kit for that.

Before doing this we should confirm that the HI0659 mutation we've made is responsible for the competence defect we see by 'backcrossing' the unmarked mutation into a clean genetic background,. This would be much easier if the mutation is closely linked to a marker we can select for. We could instead transform it into the marked HI0660 knockout and screen for loss of SpcR; in principle this wouldn't be as efficient as selection but it might be easier.

The RNA-seq experiment will be useful in other ways. Because the genome (and transcriptome) are small, we can afford to include lots of controls. At a minimum we'll do wildtype and mutant cells in log phase and after competence induction, but we might also include crp and sxy mutants, and maybe even one or more of the mysterious hypercompetence mutants of HI1133 (murE). This analysis will complement our previous microarray analysis, putting our identification of competence-regulated genes on a very solid foundation.

Could I use Pinterest to organize links to research papers?

2012-02-11T09:12:00.000-08:00

I spent much of yesterday going through piles of papers on my desk, throwing out some and sorting others into topic piles. Most of it was printouts of pdfs of research papers, which I keep mainly to remind me that the paper exists. Hype about the new social media site Pinterest is building, and I'm now wondering whether it might be something I could use to visually organize links to papers, rather like spreading them all out on the floor of my magically-expanded office.

I don't have a Pinterest account yet (you have to request an 'invitation', for which you may have to wait a week or more), but here's how I think it works: You set up 'boards' with different topics, and then add items to them by clicking on images you find on the web. This adds the image to your board, with a title and a link to the source. What I'd like to do is create boards for different components of my research (different bacteria, different proteins or functions, etc.) with pinned links to relevant research papers.

Pinterest is set up to work with images, not pdfs. Luckily, research papers almost always include figures, so if you're viewing the html version rather than the pdf version I think you should be able to pin (link to) the paper by clicking on any of its figures. The key step would be giving each link a short informative title reminding you why you wanted to remember it. Has anyone tried this?

I can't try Pinterest out for myself until I get my invitation email. In the meantime here's an image of Carl Zimmer's Pinterest page:

Back to the CIHR grant proposal

2012-02-10T18:42:00.000-08:00

We've submitted four (!) papers in the past two weeks: the arseniclife paper, now under review at Science; the RA's E. coli competence paper, submitted to PLoS One after being bounced back to us by Journal of Bacteriology, the postdoc's DNA uptake paper, submitted to PNAS Plus; and the visiting grad student's paper about Gallibacterium anatis competence, submitted this morning to Applied and Environmental Microbiology.

Our big CIHR grant proposal is due at the end of the month. This is yet another (improved) variant of the DNA uptake proposal we've submitted several times over the past few years. On those occasions it would have been a second grant, but our current grant will end in September so this time the proposal will be for the renewal of the current grant. That means its success is more important than in the past. Again we're fortunate to have a colleague critiquing our draft for us, arranged through a in-house peer-review program that used to be called HeRRO but now might be called something else.

The figure is one we'll be including in the Background section of the proposal. It shows the predicted cellular localizations of all of the proteins of the H. influenzae competence regulon, colour-coded to indicate the effect of each knockout mutation on DNA uptake.

Authorship without responsibility?

2012-02-03T06:38:00.000-08:00

I'm becoming increasingly disturbed by the behaviour of Wolfe-Simon's arseniclife coauthors. She shared the credit for the work with 11 other authors but, in the year since the tide of support turned, the senior author is the only one to have said even a word to support her or defend the work. And even he mostly says 'No comment' or 'We'll wait for the peer-reviewed responses'. All of the authors signed the Response to Comments published in early June, so I presume they stand by the work. Why then is Wolfe-Simon the only one speaking up to defend it?

David Dobbs made this point very well in a post last September on his Wired Neuron Culture blog (Arsenic is Life and the View From Nowhere):

Meanwhile, I know that part of what unsettles me about this story, regardless of how much sympathy one feels is due Wolfe-Simon (and I generally lean toward sympathy), is how both NASA and her mentors and former lab heads seem to have abandoned Wolfe-Simon. It appears they bought and fueled the bus; put bright lights and banners on it; cheered as Wolfe-Simon drove it a bit wildly honking the horn; and have now thrown her under it.

Here's the author list from the paper. Some of these people are junior members of the Oremland group, or of other research groups, but others are senior scientists with their own NASA-funded laboratories:

Felisa Wolfe-Simon: The lead author, at that time a NASA-funded postdoc in Ron Oremland's group.
Jodi Switzer Blum: A long-time member of Ron Oremland's group.
Thomas R. Kulp: At the same USGS Menlo Park laboratory as Ron Oremland; has been publishing with them and others since 2004.
Gwyneth W. Gordon: Assistant Research Scientist in Ariel Anbar's group.
Jennifer Pett-Ridge: Scientific staff member at Lawrence Livermore National Laboratory. Expertise: Environmental microbial ecology; biogeochemistry; stable isotope probes for analysis of nutrient cycling, molecular genomics of environmental microbial communities, subcellular imaging via TEM and NanoSIM.
John F. Stoltz: Director, Center for Environmental Research and Education, and Professor, Environmental Microbiology, at Duqueyne University. Expertise: microbial arsenic transformation, chromate reduction in the presence of high nitrate, community structure in modern marine stromatolites.
Samuel M. Webb: A beam line scientist at the Stanford Synchrotron Radiation Lightsource (SSRL) in the Structural Molecular Biology (SMB) program.
Peter K. Weber: Scientific staff member at Lawrence Livermore National Laboratory. Expertise: Environmental geochemistry; microbial geochemistry; elemental and isotopic tracers; salmonid migration and survival; and mass spectrometry.
Paul C. W. Davies: Director of the Beyond Center for Fundamental Concepts in Science and co-Director of the Cosmology Initiative, both at Arizona State University.
Ariel D. Anbar: Professor at Arizona State University. Expertise: environmental chemistry of bioessential and redox-sensitive transition metals, using the isotope biogeochemistry of iron, molybdenum and other “non-traditional” stable isotope systems to examine changes in metal availability through time, particularly in the Precambrian, and to develop novel isotopic biosignatures.
Ronald Oremland: Senior Scientist with the USGS Laboratory at Menlo Park. Expertise: microbial metabolism of reduced gases (e.g., methane, ethane, methyl halides, acetylene), and of toxic elements including selenium, arsenic, tellurium, mercury, and antimony.

The behaviour of these researchers suggests that they're happy to accept credit for this work (a paper in Science to list on their CVs) but unwilling to accept any responsibility for its quality. Perhaps they see their contributions as contract work—they delivered their data, were paid with authorship, and washed their hands.

Open peer review of our arseniclife submission please

2012-02-01T07:52:00.000-08:00

Our manuscript reporting the lack of arsenate in the DNA of arsenate-grown GFAJ-1 cells is now available on the arXiv server at http://arxiv.org/abs/1201.6643.

I posted it there mainly out of principle (openness is good), but it's already attracting some critical commentary. This reminded me that one of the main purposes of the arXiv is to encourage pre-publication discussion of research. This is open peer review!

So please post your comments on our manuscript here. To get things started, here are the comments already made:

NotAnAstrobiologistJan 31, 2012 09:42 PM

As I understand it, Figure S1 has error bars which represent the standard deviation of ion counts for independent purifications of the same DNA sample, characterizing the variance across purifications.

Why use the standard deviation in this case where your sample size=2? Using the two actual values would make more sense to me (estimating the distribution in this case obfuscates the underlying data, as you've irreversibly "reduced" two observed values to two statistical estimates). I think it makes more sense to show the actual observations, or do (at least) three experiments...

Later:

FWIW to make sure I wasn't making it up (I've seen error bars on small n estimates before), note the line:

"However, if n is very small (for example n = 3), rather than showing error bars and statistics, it is better to simply plot the individual data points."

Error bars in experimental biology
http://jcb.rupress.org/content/177/1/7.full

Ryan FFeb 1, 2012 06:19 AM

Or have the error bars indicate the range rather than standard deviation?

The #arseniclife manuscript has been submitted!

2012-01-30T11:25:00.001-08:00

We've posted the manuscript on the public arXiv.org server. You can download the full pdf, including all the supplementary data, at http://arxiv.org/abs/1201.6643.

Lists of Elsevier journals to boycott

2012-01-30T03:11:00.000-08:00

Readers of this blog probably already know that there's a call out to boycott journals published by Elsevier because of their anti-scientific publishing practices. Initially researchers were signing a pledge to not contribute to Elsevier's activities, by refusing to publish in, referee for, or do editorial work for any Elsevier journal. I've signed this (at The Cost of Knowledge), and you should too.

Jon Eisen has now expanded this, asking that researchers also refrain from promoting work published in Elsevier journals (Boycotting Elsevier is not enough - time to make them invisible). Don't write blog posts about them, don't choose them for journal club, don't even cite them if a reasonable alternative is available (he didn't say that last one, but I do).

This is all very well in principle, but if you're like me you have only a very fuzzy idea of which journals in your field are part of the Elsevier empire. The information is available for all fields on Elsevier's web site, but I thought I'd do my bit by listing here the journals in my field. But then I saw how many there are! Of course my 'field' is an unusual overlap of genetics and molecular biology and microbiology and evolutionary biology, but still. Here are a few you might recognize:

Elsevier's list (148 journals): Molecular Biology, Cellular Biology and Genetics:

Elsevier's list (94 journals): Microbiology and Virology

Elsevier doesn't have a list for Evolution or any related topic, so I did a search. Although that identified 179 journals, most are not focused on evolution. Here's a link to the first 25 on the list.

ArXiv submission?

2012-01-29T13:52:00.000-08:00

I'd like to put our arseniclife submission to Science onto the arXiv server so that anyone who's interested can read it. Not many biologists use arXiv (it's mainly a physics thing) but it's a very convenient place to post manuscripts and other documents. And its use by physicists provides a great precedent for open science, because manuscripts are posted there and submitted for formal publication in peer-reviewed journals.

However, I'd like to first find out whether Science has any policy about arXiv pre-publication. Their Instructions to Authors say:

Distribution on the Internet may be considered prior publication and may compromise the originality of the paper as a submission to Science. Please contact the editors with questions regarding allowable postings.

Has anyone had direct experience with this? I think I'd better send out a tweet...

Brief update

2012-01-28T11:24:00.000-08:00

Things are progressing much faster than usual - this will be an epic week for paper-submitting!

The Research Associate submitted her manuscript about natural competence in E. coli a few days ago, to the Journal of Bacteriology. She's done a mass of work showing that a wide range of E. coli strains (including the full ECOR collection) are not naturally transformable even when their competence regulons are induced by artificial expression of Sxy from a high-copy plasmid. But a bit of transformation does happen if recombination functions are also artificially provided by inducing the lambda 'recombineering' genes. So the competence regulon does encode a functional DNA uptake machinery. We don't know why it's so inefficient compared to those of other bacteria, though we make a few suggestions.

The arseniclife analyses have all been replicated and the manuscript is almost ready for submission to Science as a Report. We're aiming for Monday - the grad student and his supervisor are still polishing up their figures.

After what seems like an eternity of wrestling with his DNA uptake specificity data, the analyses, and the interpretations, the post-doc and I now agree that we have an excellent manuscript that will be ready to submit to PNAS within a few days.

A manuscript by a visiting grad student from a few years ago is also going to be submitted within the next few days. It describes her investigations into competence of a relative of Haemophilus influenze, the poultry pathogen Gallibacterium anatis. We're listed as authors because some of the work was done in our lab, and because we've contributed quite a bit to the analysis and writing.

And finally, my article about how the teaching of introductory genetics needs to change is just about ready to send to PLoS Biology!

Sorry for lack of posts...

2012-01-26T17:50:00.000-08:00

We're busy finishing the Science/arseniclife paper, and the postdoc's uptake paper, and the RA's E. coli competence paper (submitted!), and an old visitor's competence paper, and my article about teaching genetics....

Sudsy gel

2012-01-22T17:23:00.001-08:00

Why did I put SDS into the buffer of this agarose gel before I loaded it? So the DNA from the lysed cells wouldn't rise up out of the wells and spread out over the surface of the gel buffer, of course!

I'll tell you more tomorrow, if my experiment works out

The Discussion for the post-doc's DNA uptake paper

2012-01-20T19:14:00.000-08:00

The post-doc and I have been struggling, independently and together, to create a good Discussion section for his paper on the sequence specificity of DNA uptake. We have lots of things we could write about, but many of them aren't well connected to each other or to what the paper is about. But now that I've done some good work on the end of the Results, I think I've finally come up with a Discussion that might work.

The Results ends with the analysis of possible interactions between bases at different positions in the uptake signal sequence motif he derived. We motivate this analysis as a possible explanation of the discrepancy between his uptake sequence motif and the one I derived years ago for the uptake sequences in the H. influenzae genome. I'm reproducing the two motifs below and below them his figure of his interaction analysis.

He's now done an uptake experiment that validates (confirms the predictions of) the interaction analysis. It shows that having mutations at two interacting positions (positions 4 and 11, I think) does indeed reduce DNA uptake much more strongly than predicted by the effect of each mutation singly. This motivated me to clarify for myself the implications of the interaction analysis.

The diagram is at the top of this post. The center four positions of the core (left segment) are greyed out, because their effect on uptake is so strong that we can't make confident inferences about their interactions with other positions. The black brackets above each segment indicates that all of the bracketed positions participate in interactions with all the positions in the other bracketed segments, as indicated by the blue arrows. However the positions within a single bracketed segments do not interact with each other, unlike the minor covariation interactions (figure below) we found long ago between adjacent positions in the genomic USS sequences (pdf of the paper here).

Anyway, back to the Discussion...

First we can explain how the interaction analysis nicely reinforces the hypothesis that the uptake sequences in the genome are there as a direct and unselected molecular-drive consequence of the bias of the uptake machinery. This is an 'exception that proves the rule' situation, where the initial finding that the simple uptake-bias motif didn't match the genomic USS motif created doubt about the hypothesis, and the subsequent demonstration that interactions explain the discrepancy increased our confidence in it.

Then we can say that this leaves only the uptake bias itself in need of an explanation, and that we propose that it exists as part of a solution to the mechanistic problem of getting stiff, highly charged DNA molecules through the narrow secretin pore. Because cells efficiently take up closed circular DNAs we know that uptake doesn't usually initiate at a fragment end, but must initiate internally on DNA fragments (see this very old post). We hypothesize that the uptake bias favours sequences that are readily kinked, and that this kinking occurs mainly as consequence of interactions between the uptake sequence and mutually-interacting proteins of the uptake machinery (the uptake motif is itself only slightly bent, at the T-tracts). One reason to think that proteins mediate the interactions is that adjacent positions don't interact with each other.

Perhaps we can here pose a specific model of what parts of the uptake sequence interact with what parts of the machinery... This should take into account that the T-tracts interact with the core positions but not with each other.

Finally we can discuss the known or possible uptake biases of other species. First the other Pasteurellaceae, then the Neisserias, and finally bacteria where uptake bias may have been overlooked.

Growth of GFAJ-1 under phosphate limitation (correction)

2012-01-20T16:09:00.000-08:00

Erika Check Hayden's otherwise-excellent Nature News report on our work contained one error, the statement that "Redfield was unable to grow any cells without adding a small amount of phosphorus".

Here's the email I had sent her in response to an earlier query about phosphorus concentrations:

Hi Erika,

The amount of phosphate in the medium used by Wolfe-Simon et al for their published growth analysis is indeed uncertain. Their ICP-MS analysis found that most of their media preparations contained 3-4 µM phosphorus, but one batch contained <0.3 µM and a solution containing only the AML60-medium salts had 7.8 µM. Because we don't know which batch was used for the results in their Figure 1, 3-4 µM is a good estimate of the phosphorus contamination, but the actual amount could have been substantially lower or higher.

My cells did grow in medium with no added phosphorus*, to about 5 x 10^6 cells/ml. This is about 1/4 of the density reached by GFAJ-1 in Wolfe-Simon et al's '-P/+As' medium. Adding 3 µM phosphorus to my medium increased GFAJ-1 growth fourfold, to the same density as reported in Wolfe-Simon et al's experiments. Simple algebra thus suggests that my unsupplemented medium contained about 1 µM phosphorus. The correspondence of the cell densities reached in my supplemented (3 µM) and their unsupplemented medium supports the estimate of 3-4 µM contaminating phosphorus in their medium.

My cells, like theirs, were clearly phosphorus-limited, because they grew to much higher densities when additional phosphorus was provided (see my recent RRResearch post and their Fig. 1).

I think this is the best that can be done, since Wolfe-Simon et al. apparently did not keep good enough records to determine the actual phosphorus concentration of the medium they used for their reported experiments.

Hope this helps,

Rosie

*The initial growth problem was not due to a lack of phosphorus but to the need for an amino acid, which I solved by supplementing the medium with a small amount of glutamate.

Not me!

2012-01-17T16:25:00.000-08:00

GFAJ-1 growth curves in limiting phosphate

2012-01-17T11:43:00.000-08:00

The BioScreen is a wonderful time-saver. Over the weekend it did growth curves using media with 9 different concentrations of phosphate, each with 10 replicates, taking readings every 20 minutes for 46 hr!

This data tells me that my choice of 3 µM added phosphate was good; it gives about four times as much growth as no added phosphate, and twice as much as 1 µM, so the unsupplemented medium probably has about 1 µM contaminating phosphate.

The big surprise is that cells reach higher densities with a moderate amount of phosphate (70 µM) than they do with 250 µM or with the 1500 µM used by Wolfe-Simon et al. I don't think this has any serious implications for our analysis.

I was also surprised to see that the cultures with the higher amounts of phosphate were still growing at the end of the time course. I'm going to replicate these results with another time course, and this time I'll run it for longer (3 days? 4 days?).

The CsCl/mass spectrometry data

2012-01-16T19:53:00.000-08:00

Here's the figure the collaborating grad student sent, showing his LC-MS analysis results of two DNA samples from the first set of GFAJ-1 preparations I sent him.

Each data point is a fraction from one of the CsCl gradients he fractionated the two GFAJ-1 DNA samples on (one for the -As/-P DNA and one for the +As/-P DNA). The -P condition is actually 3 µM added phosphate - this gives growth to approximately the same density as Wolfe-Simon et al's '-P' condition.

The lines with the solid symbols show the amount of DNA in each fraction - these each show a nice DNA peak at around the 800 µl position in the gradient.

The lines with open symbols show the amount of arsenate in each of these fractions - these lines are hard to see because they're sitting right on top of the X-axis (yes, that means that the amounts of arsenate detected are ~ zero 'ion counts'). The real values aren't necessarily zero, but they're below the detection limit for this experiment.

The dashed line shows the amount of arsenate that should have been detected if 4% of the phosphate in the DNA had been replaced by arsenate, as predicted by Wolfe-Simon et al's gel analysis (data in their Table S2).

The second graph shows his standard curve for arsenate detection.

Academic publishing gets even sleazier

2012-01-15T08:26:00.000-08:00

An email from Scientific and Academic Publishing:

Dear Rosemary J. Redfield,

This is Scientific & Academic Publishing, USA. Nice to get your information from the journal PLOS Pathogens and also happy to pass on our regards to you from the editorial department of SAP.

We've finished reading the abstract of your paper Transformation of Natural Genetic Variation into Haemophilus Influenzae Genomes and will recommend it to our editors. If you are interested in our journals and want to publish it on our journals, please extend this paper and describe your latest research achievements and send it to us by our online submission system (http://www.manuscriptsystem.com). All manuscripts submitted will be considered for publication.

If this paper has been published, we also welcome you to submit other papers to us.

Welcome to visit our website at http://www.sapub.org.

In the second paragraph they seem to be first saying they'll recommend my already-published paper to their editors (for the editors to do what, read it with admiration?), and then asking me to add a bit of new material to it and submit it to them for publication. This reeks of self-plagiarization. But in the next sentence they ask for other papers instead.

Who are these guys? Their web site lists an impressive 133 journal titles. But most of the ones I clicked on are nonexistent - they have some Editorial Board members but no Editor in Chief or ISBN number, and haven't published any papers. Only one (The International Journal of Plant Research) had 'published' any papers, and these each had only Abstract and reference list- the body of the paper was apparently 'coming soon'. Perhaps this is to be expected, given that this journal too lacks an Editor in Chief. It may lack editors entirely - authors are instructed that they must format the html links for the references they cite, a function normally done by a journal's copy editors.

Their office is in California, so they're not a third-world effort. I couldn't find any information about publication charges at all, but I don't suppose they're just doing this for the glory.

Hmm, the International Journal of Genetic Engineering needs an Editor in Chief - that would look good on my CV. All I need to do is check the boxes on the handy application form they provide!

Here's the gel photo

2012-01-14T17:43:00.000-08:00

These DNAs were all stored in the fridge (4 °C) in aqueous solution (10 mM Tris 1 mM EDTA pH 8.0) for two months before this gel was run. The DNAs in the 'ss' lanes were heated to 95°C for 10 min before loading to separate the strands and reveal the effects of any single-strand breaks.

These DNAs show no sign of degradation; compare to the original photo here. In particular, the DNA fragments from cells grown with limiting phosphate and 40 mM arsenate are actually slightly longer than the fragments from cells grown with limiting phosphate and no arsenate. (I don't think this difference is significant; the important point is that the fragments aren't any shorter.)

Because these large fragments typically migrate at the resolving limit of the gel, all I can say with confidence is that the fragments in all four preps are all significantly larger than 30 kb. This is the size range we expect for chromosomal DNA in a normal DNA prep. I don't have size standards for single-stranded DNA (I should have heated the lambda fragments but forgot to) so all I can say about the length distribution of single strands is that the four preps are all very similar.

This result tells is that DNAs from arsenate-grown cells are not undergoing degradation in storage due to slow hydrolysis of arsenate diester bonds in the DNA backbone, as suggested by an earlier anonymous commenter.

Generating final data for the #arseniclife paper

2012-01-14T05:11:00.000-08:00

1. Cells for new DNA preps: For the replicate DNA preps (for the replicate LC-MS analysis), yesterday I inoculated GFAJ-1 cells into two 50 ml cultures in AML60 medium with 1500 µM PO4, with and without 40 mM AsO4, and into two 500 ml cultures on AML60 medium with 3 µM PO4, with and without 40 mM AsO4. Most of these cultures are growing nicely, so tomorrow I think I'll have enough cells for the DNA preps. Well, the 1500 µMp 40 mM As culture isn't growing at all, but I don't think we need to replicate this one anyway. I need to get at least 50 µg of DNA from each prep, to give the grad student enough for his CsCl gradients. Last time one of the cultures (3 µM PO4, no AsO4) wasn't dense enough to give me the DNA I needed, but so far it looks as dense (or not-dense) as the parallel culture with AsO4. I'll prep the DNA today and if I don't have enough I'll just set up more cultures. I'd be able to prep the DNA from them on Sunday, so still would have the DNAs ready to send on Monday.

2. Troll-suggested control: I've run the gel of the two-month-old DNAs from cells growth with and without arsenic, both native and denatured, and there's no difference in fragment length, with all double-stranded fragments being at least 30 kb in length. So there's no evidence of arsenic-bond strand breakage during long-term storage at 4 °C. I'll post a gel photo later (the image I saved isn't right).

3. Presentable growth curves: A lab in our research cluster has a BioScreen incubator/plate reader I can use to automate my growth curves. But the test cultures I set up in an ordinary microtiter plate aren't growing consistently, so I'll have to mess around a bit before I can do the growth curves.

Writing the #arseniclife paper

2012-01-11T14:13:00.000-08:00

The grad student working on the mass-spectrometry analysis of GFAJ-1 DNA is still making sure his results meet his high standards, but as soon as they are ready he'll send them to me and I'll post them here. In the meantime, since he and his supervisors have concluded that the DNA contains no arsenic, we've started writing our paper. We're going to submit it to Science as a Brevia. These are very short peer-reviewed articles (one page, one figure), which we think suits this work very well.

But first we need to replicate our results. My plan is to generate some detailed growth curves for cultures with various levels of phosphate, with and without 40 mM arsenate. For this I'll use a BioScreen machine that belongs to a neighbouring lab. This machine automates collection of optical density data from cultures growing in wells of 100-well plates. I'll also grow big batches of cells for new DNA preps, using the same media and culture conditions as before.

This should only take a few days, and I hope to have the DNAs ready to send to my collaborators on Monday.

Two steps forward, one step back (the postdoc's uptake bias paper)

2012-01-11T08:54:00.000-08:00

The postdoc's manuscript on uptake bias is inching towards completion. He's added most of the references and updated the figures, and we've only discovered one new analysis that needed to be done. But including this analysis at the right place in the Results makes writing the rest of the Results a lot more straightforward, so we're ahead of the game.

What is this analysis? Removing, from our dataset of 10^7 sequence reads of DNA fragments that the competent cells took up, some sequences that may have been interpreted incorrectly. The incorrect interpretation happens because the sequence responsible for their uptake isn't correctly aligned in our analysis. Here's a figure explaining the problem:

The top sequence is the consensus of the fragment we used. The lower-case bases at each end were not degenerate and function as controls. The first step in the analysis was to align each sequence read to this consensus at its left end, and below the consensus we see three correctly aligned reads, with their core uptake sequence indicated by the yellow arrows.

Below these are two reads that were misaligned because they contained either an insertion or a deletion of a single base. We think these insertions and deletions arose during synthesis of the pool of degenerate fragments. Although these fragments still contain good uptake sequences (red arrows), the incorrect alignment doesn't recognize this. Instead, the fragments appear to have been taken up despite having very poor agreement with the consensus.

Below these misaligned reads is a sequence that is correctly aligned but that contains a second match to the core consensus, indicated by the green arrow. This second match was created by several changes downstream of the consensus uptake sequences, but it isn't recognized by the analysis because it is out of alignment and, in this case, in the other orientation. The presence of two uptake sequences means that we can't attribute their uptake to the one sequence that's correctly aligned.

Sequences with these artefacts couldn't be removed from the dataset before the original analysis, because they couldn't be identified until we were able to score each fragment for matches to the 'uptake motif' that the initial analysis produced. Now that we've identified them, we can consider whether they would have confounded any of the analyses.

The main concern is the reads with insertions or deletions. Because the initial filtering required that the 10 control bases all be perfectly matched, most of these were removed, and the 10^7 recovered reads we analyzed only included about 1500 with insertions or deletions that misaligned the core. That's too few to have misled the initial analysis, but it is a concern for the analyses of possible contamination and sequencing errors, and for the analysis of interaction effects. The postdoc has now finished checking for effects on the interaction analysis (none) and still needs to check for contamination and error effects.

A troll raises a semi-valid concern

2012-01-09T22:02:00.000-08:00

An anonymous troll posted this comment on a previous post:

im curious about how much time the DNA you prepared has spent in solution since you prepped it — supposing there were C-Ar bonds in the DNA at the time of lysis, has so much time passed now that, given the hydrolysis rates that were experimentally determined and recently reported in this JACS communication (offensive link deleted), I'm worried that theres no hope of seeing positive signal even if there was one to begin with. Any thoughts about this issue? Perhaps keeping the DNA stored as a dry pellet and avoiding any encounters with water until the last minute would be the way to go.

First, a few corrections: Arsenic is As, not Ar. The DNA backbone bonds are diester bonds, so the As is bound to oxygen (O), not carbon (C). It's of course not possible to purify DNA while avoiding any encounters with water.

It's indeed theoretically possible that the GFAJ-1 DNA from arsenic-grown cells originally contained As in its backbone, and that the As bonds were destabilized once the DNA was purified away from the proteins and other cellular components. If the chemists are correct, the As bonds would all hydrolyze within less than a second. The AsO4 liberated from the DNA would then be seen just as background in the CsCl gradient, or lost entirely in subsequent purification steps.

We would have to assume that the GFAJ-1 cells contain some powerful unknown-to-science DNA-binding proteins or other structures that stabilize the As bonds in the aqueous environment of the cell. These proteins would be removed during the initial DNA purification steps (SDS-lysis and phenol-extraction). Wolfe-Simon et al. did not report taking any precautions to prevent hydrolysis, so if their DNA prep really contained As bonds these must have been stable for at least the day it would have taken them to do the initial extractions, run the agarose gel, and cut out the DNA-containing gel slices. We don't need to count the time needed to send the gel slices to the LLNL lab at Livermore for Nano-SIMS analysis because the entire slices were analyzed (the DNA was not purified away from them).

Loss of As from the backbone would change the structure of the DNA. It would now be As-free, but would contain a single-strand break at each site that previously had an As. If a substantial fraction of the backbone was As, the DNA would be extensively degraded.

One way to check for hydrolysis of As bonds would be to run the DNAs in a gel, both intact and after separating the strands by boiling. Normal DNA is stable for months, so we could compare the fragment lengths of the DNAs from As-grown and P-grown cells, both immediately after purification and after extended storage in aqueous solution. Here's a diagram of what we'd expect to see in an agarose gel. The upper orange band in the lefthand lanes is the long fragments of double-stranded DNA present in my DNA preps immediately after purification (see gel photo here), and the broader bands on the right are how single-stranded DNA would appear.

Later: Comment send by email:

Since the cell’s interior is aqueous, then it would seem reasonable that there has to be something preventing the spontaneous hydrolysis in vivo. As you point out, some form of stabilizing protein would seem the most likely candidate. If this protein is going to work, then it could (for the purposes of this argument) remain attached throughout the purification process. If so, then you might find As in the purified DNA, and should also find traces of protein. I don’t know if this would be enough to alter the 260:280 ratio, but DNAse digestion followed by SDS-PAGE might detect something. Definitely a hotdog experiment, but once the DNA is purified, not that difficult.

SDS-phenol extractions are pretty harsh; for a protein to remain with the DNA it would need to have been covalently bound, which would certainly have complicated such cellular processes as DNA replication and transcription. And if the DNA contained a significant amount of As, the DNA would then contain enough protein that it wouldn't migrate properly in the gel but would stick as a blob of gunk in the well at the top of the gel. It also would band differently in a CsCl gradient.

How dumb do (a couple of) our students think we are?

2012-01-08T16:52:00.000-08:00

Email I just received from a couple of undergraduates:

HI Dr.Rosie Redfield,

Here's a chance to represent UBC's Zoology Science Department.

The annual Science Week Events Committee , organized by the Science Undergraduate Society, would like to invite you to join our event on Thursday, January 26th, 2012 at 12:30-1:45.

As you may know, Science Week, is a week-long (form January 23rd - January 27th), multi-events celebration which allows students to show off their UBC pride while rewarding them for their first term achievements. So far we have jell-O wrestling, jeopardy, and a scavenger hunt. Although, we realized something was missing...(*1)

This year, we have added a new event to our venue, called the "Professor Pageant". UBC Professors, like yourself, will be participating in the pageant and showcasing their popularity, attitude and talents (*2). Only one competitor will come out on top, however, each participant will go home with a special customized award.

We would like to invite you to join us because you are deemed awesome (yes, awesome) by an overwhelming consensus among students (*3). This is an opportunity that you do not want to miss! (*4)

We would love to hear from you and provide more details. Also, if you would like to suggest any of your colleagues (open to all faculties), please provide their name so we could can invite them accordingly!

Sincerely

(Names withheld to protect the naive.)

(*1): Maybe it's "science"?

(*2): Ooh, why didn't anyone tell me that's what I'm valued for?

(*3) Somehow these students neglected to complete their evaluations of my teaching.

(*4) Oh yes I do!