Should Darwin be an 'ism'?

On Tuesday evening I'm leading a Cafe Scientifique discussion on the topic Should Darwin be an 'ism'? I chose this topic as something that a broad range of people would be interested in and have ideas about, but I need to do some reading and thinking first.  Luckily the discussions take place in a local pub (The Railway Club, 579 Dunsmuir, 7:30pm, in case you're interested), and the atmosphere is very informal.

What will I read?  Carl Safina had a very relevant article in the New York Times last month (Darwinism must die so that evolution may live, Feb. 9), which I need to read carefully.  When it came out I didn't take the time to read it properly because I expected to agree with everything it said.  But I also need to read a bit more history of the use of the term Darwinism, maybe in Ernst Mayr's The Growth of Biological Thought. 

What do I think?  Maybe biologists started referring to evolutionary theory as Darwinism as a way to give credit to a truly exemplary scientist.  But now the creationists are turning this against us, claiming that we 'worship Darwin like a god', and that any evidence that Darwin made any error is evidence that evolutionary theory is wrong.  Using the term Darwinism also lets people put evolutionary biology in with a pile of what are now largely discredited ideologies and belief systems (Marxism, Raelism, Freudian psychology, etc.).

Unfortunately the cat is out of the bag.  Getting evolutionary biologists to forego using Darwinism will be easy, but re-educating the general public will be much harder.  The real problem is still that the creationists are much better publicists than we are, and they are determined to keep the public believing that evolutionary biology is synonymous with Darwinism.

Open access at the American Society for Microbiology annual general meeting

In May I'll be part of a panel discussion on open-access publishing, at the big General Meeting of the American Society for Microbiology.  The other participants are 'professional experts': Jon Eisen, Academic Editor in Chief, PLoS; Sam Kaplan, Chair of the ASM Publications Board (ASM publishes about a dozen journals and many books); and Joe Deken of the California Institute for Telecommunications and Information Technology.  I guess what I'll bring to the table is the perspective of the ordinary scientist trying to do what's right.

Writing

In addition to How to Write a Lot, I've been re-reading a little book on writing by Joseph Williams, Style, the Basics of Clarity and Grace.  This wonderful book is mainly about how to write sentences that are easy to read and understand, something all scientists strive for but few of us achieve.

One reason scientific sentences are often hard to follow is called 'nominalization'.  That's when an action is described by a noun rather than a verb.  For example, instead of writing 'the cell divided' we might write 'cell division occurred'.  I'm building my ability to avoid this by going through the manuscript I'm revising, rewriting sentences that suffer excessively from nominalization.  I don't have to search for these sentences, almost every sentence has one or more nominalized actions in it.

Here's an all-too-typical example:  "In E. coli, the dramatic reduction in growth and eventual cell death caused by sxy overexpression made it impossible to test whether sxy induction produces the typical ‘natural competence’ phenotype of high-efficiency transformation with linear chromosomal DNA."  It's a perfectly OK sentence, no grammar or syntax errors, but it's still a bit of an effort to read.  Can I improve it by replacing some of the nominalizations (reduction, overexpression, induction, transformation) with verbs?  

Yes I can.  "We could not test whether inducing sxy causes E. coli cells to become naturally competent and efficiently transform with chromosomal DNA, because when cells overexpress sxy their growth rate slows and they eventually die."

Small steps

Prompted by the How to Write a Lot book, I'm trying to spend half an hour on a scholarly-writing task before I get up, each morning that I don't have to be somewhere early (i.e. not at the gym by 8:00am).  As a result of this, I've nearly finished the first draft of my short essay for the ASM evolution book.  Only about 3 paragraphs still to go, and I know what they're going to say!

Yesterday I started reading the revised manuscript about Sxy in E. coli.  It still needs a fair bit of rewriting work, but maybe the post-doc and I will have enough time for sitting-down-together-and-revising that we can still get it out by the end of the week.

Manuscript work

The end of term is approaching so I can see the light at the end of the teaching tunnel (mixed metaphor?).  Here's a list of the manuscript-related tasks on my plate:

Informal chapter for the feitschrift for John Roth:  The first draft is in the hands of the editors, who I hope will soon give me feedback on how to improve it.  The post-doc and undergrad also have it - I haven't had any feedback from them either.

Short essay for the ASM popular science book on Darwin and microbial evolution:  I'm working on this.  I need 2000 words and have about 1500.  It's turning into a nice discussion of how we can study natural selection in bacteria.  I should soon have a list of all the other authors and their topics, which will help me integrate mine.  I just reread the email invitation, and now realize that I'm supposed to include personal stuff about me as a scientist - maybe I will, maybe it won't fit.

Manuscript about regulation by E. coli Sxy:  This was gently rejected by J. Bacteriology (with the possibility of resubmission).  The post-doc first-author has now rewritten it for resubmission, with input from the former post-doc other-author, and she's now passed it on to me.  If it's OK we'll submit it this week. 

Manuscript on co-evolution of uptake sequences and proteomes:  This has been provisionally accepted by Genome Biology and Evolution.  I asked the bioinformatician coauthor for feedback -she sent me a short email with some questions I haven't responded to.  So the first step is to respond to her questions (well, after I re-read the reviewers' comments).  I'm hoping we won't need to do any substantial new work.

Manuscript on student writing and learning:  This has been languishing since my teaching-fellow post-docs left town.  It's nearly finished so I should get it done.

Manuscript on the perl model of uptake sequence evolution:  As I recall, this needs a bit more computer-simulation work and quite a bit more writing.  The post-doc senior author has moved to Toronto, but we should still be able to get this done.

Manuscript on the phylogeny of H. influenzae strains and competence:  This is the work of this same post-doc.  The manuscript was provisionally accepted, but with requests for substantial additional work.  Based on her past performance I'm confident that she will get this done, but I should touch bases with her about it.

Can that be all?  

progress (?) on the ligase puzzle

The NHEJ expert said that he thought the periplasmic assignment of the H. influenzae ATP DNA ligase must be an error.  I was discussion the ligases with a colleague who works on Campylobacter (which also has one of these ligases) and she suggested I try running the sequences through the program PSORT-b, which is particularly good with bacterial proteins.  

PSRT-b could not assign a high-probability location to most of the ligases I tried, suggesting that the HMM method used by TIGR's database may be overconfident.  I was also surprised to find that its BLAST search pulled up some NAD-dependent ligases as matches to the ATP-dependent ligase sequences I tried.  I had been thinking that the two families had very dissimilar sequences, but maybe I'm wrong in that. 

The possibility that these ATP-dependent ligases act in the cytoplasm is interesting, as the competence-induction of the H. influenzae one may mean that it contributes to the postulated replication-arrest problem rather than to DNA uptake.

That periplasmic ligase

Yesterday I talked to Tom Silhavy about the periplasmic ATP-dependent DNA ligase that's co-induced with H. influenzae DNA-uptake genes (see old blog post here). He hadn't heard of this and was adamant that there is no ATP in the periplasm. So I did some more poking around.

I found papers about bacterial ATP-dependent ligases that function in 'non-homologous end joining' (NHEJ) reactions - these serve as last-resort repair mechanisms for double-strand DNA breaks that can't find a homologous template to use for repair. I emailed the author of a review, asking if the H. influenzae ligase belonged in this category. (He turned out to also be the person who had done the biochemical characterization of the H. influenzae ligase!)

He said that H. influenzae doesn't have the other NHEJ genes Ku and LigD, so it probably can't do NHEJ. I suspect the H. influenzae protein is in a different category of ligase, because a BLAST search with the H. influenzae ligase doesn't find known NHEJ ligases.

He also asked why I think it's targeted to the periplasm. At first I thought he meant, what do I think is the reason it's target to the periplasm, so I explained that I don't know. But then I realized he might be asking what is the reason I think it's targeted to the periplasm. I couldn't remember so I looked at it and its homologs using TIGR's HMM (hidden Markov model) location analysis function - this says that the H. influenzae protein and the four homologs I checked (Neisseria, Campylobacter, Shewanella and Thiomicrosomethingorother) all have a high probability of being periplasmic, with a single strong transmembrane domain close to the N-terminus. Tim VanWagoner, who also worked on the H. influenzae gene, also wrote that its Vibrio homolog is predicted to be periplasmic. Tom Silhavy had wondered if the apparent signal sequence might be an annotation error (wrong start site?), but this is very unlikely to be the case for all the homologs, so the odds are very high that these really are periplasmic.

I mentioned to Tom my idea that the ligase might be exported to the periplasm with an ATP already bound (the purified protein has its ATP covalently bound, ready for action). He said that, if that were the case, the protein would have to be exported by the Tat (twin argine translocation)system, because that's the only export system that can handle folded proteins. Luckily there's now a TatFind server, so I pasted in the various protein sequences, all of which had no recognizable TAT site in their first 35 aas.

How peculiar... We must be overlooking something important...

outer membrane issues

Later this morning I'll be meeting with Tom Silhavy, who's visiting to give the Microbiology seminar today.  He's an expert on outer membrane biogenesis, so what might I ask him about?

In the context of the development of competence, there's the timing issue.  How long should it take H. influenzae to assemble its DNA-uptake machinery once the genes have been turned on? We traditionally allow 100 minutes from transfer to starvation medium.  The microarray analysis showed that under these conditions gene expression is higher at 30 minutes than at 10 minutes.  Addition of cAMP to non-starved cells induced competence with a peak at 45 minutes.  How much of this time is needed for assembling DNA uptake complexes in the membranes?  What other factors might contribute to a lag?  Should E. coli be faster?

Another issue Tom's interested in is energy sources for periplasmic and outer membrane processes.  He might have some insight into the periplasmic ATP-dependent ligase that's co-induced with H. influenzae competence genes.  Where might it get its energy and what might it be contributing to uptake.

He might also have ideas about the "getting stiff DNA across the outer membrane without a free end" problem.  How flexible is the outer membrane to being pushed around?  And what about the cell wall - is it an obstacle we should worry about?

Should Darwin be an 'ism'?

In a few weeks I'm leading a Cafe Scientifique discussion on the topic "Should Darwin be an 'ism'?" I promised to provide a short abstract, so here goes:

Darwin's place in modern biology is unusually personal. When The Origin of Species was first published, biologists readily accepted the publicly controversial idea that all modern life evolved from simpler organisms. But they were dubious of natural selection's role in adaptation, and 'Darwinism' competed with 'Lamarckism' and then ''Mendelism' until the genetic basis of inheritance became clear in the 1930s. Since then many biologists have invoked Darwin whenever they spoke of natural selection, perhaps to make up for our original skepticism. But creationists are now turning this against us, claiming that evolution is nothing but Darwin-worship. Is it time to push Darwin into the closet?

ECOR strains

Yesterday I was filling in the form to apply for a permit to import pathogenic bacteria, so we could get the 'ECOR' set of E. coli strains. This is a set of 72 different E. coli strains from many different human and animal sources, chosen by Howard Ochman and Bob Selander to represent the diversity of this species.

Since Ochman and Selander's original analysis (1984) they've been examined for many different genotypes and phenotypes. The group that maintains the strains has a long list of papers describing work on them, but it only goes to 2001. So just now I did a Google Scholar search for 'ECOR collection' and one of the top hits was a paper by a UBC colleague, Julian Davies, describing these strains' repertoire of antibiotic resistance genes carried on integrons.

So I just emailed Julian. If he already has these strains, we won't have to bother importing them!

Experiments with stalled replication forks?

We think (I think) bacteria turn on their 'competence' genes because they are running out of deoxynucleotides for DNA synthesis. Part of this adaptive response is taking up DNA (an excellent dietary source of deoxynucleotides) and part of it is other changes that help cells cope with problems that arise when DNA replication is interrupted.

If I'm right, then cells with their competence genes already on might be better able to survive interruption of DNA replication. How can we test this? Are there antibiotics that block DNA replication, that can be used to create a transient block and then washed out? What about temperature-sensitive (ts) mutations in DNA replication genes? This might best be done in E. coli, not H. influenzae, because ts mutations don't work well in the latter ( it's intrinsically sensitive to minor shifts in temperature). E. coli also has a fine collection of already characterized ts mutations, and we now are able to artificially induce its CRP-S (competence) regulon by putting E. coli sxy on an inducible plasmid.

Manuscript progress

I've known for several weeks that one of the manuscripts we submitted before Christmas has been accepted, and this morning I got a 'provisional acceptance' email about another.  This latter manuscript is the one about the impact of uptake sequences on the evolution of bacterial proteomes.  The work was started more than 10 years ago, so it will be great to get this finally off my plate.

The reviewers were generally positive but they did suggest quite a few new analyses.  I'll have to consult with my bioinformatics co-author to decide which of these we might reasonably undertake.  There'll be some rewriting too of course - parts that seemed very clear to me were not so clear to the reviewers.

p.s.  Thanks to commenter (commentor?) Phagenista (great name) for the information about where to get the ECOR collection.  I've emailed them to ask about shipping to Canada - I think our strain import permit may still be valid.

DH5alpha is mutant in recA

The post-doc sent me a list she's compiled of the experiments we're planning and the genotypes of the E. coli strains we'll use.  So far I've just glanced at them, but I noticed that one of the strains used for recombination is recA.  This means that the protein responsible for almost all homologous recombination is missing, so recombination shouldn't happen!  We'll have to get this sorted out asap.

A new leaf?

I've been reading a little book called "How to write a lot" and I'm now abashed at how little I've been writing in this blog.  Nothing since December!  (Actually I have been writing in it lately, just not taking the final step of posting, because I find the blog format helps me to work on a book chapter that needs a conversational writing style.)  I'm going to get back to posting something every day, no matter how minor it has to be.

Yesterday the post-doc (I'm temporarily down to one, but a new one arrives next month!) presented some exciting data (I won't describe it until it's a bit more solid) that can be expanded by work with some old E. coli strains.  So I promised to draw up a detailed outline, spelling out the strains and their genotypes and how we'd use them.  

And we need to order the 'ECOR' collection of E. coli strains, and maybe some other strains that were recommended by a commenter on this blog a couple of years ago.  The ECOR collection is a set of a couple of dozen strains from very diverse human and animal sources - I don't yet know who we'd get them from.

Now that's a time course!

I tested the effect of allowing the cells time to express their new kanamycin-resistance gene, and found that it didn't matter at all. But everything seemed to be behaving well, so I went ahead and repeated the big time course experiment anyway. And it worked very nicely this time.

The top graph shows how the cultures grew under the different treatments. BHI is the rich medium, and they grew nicely in it. Adding 1mM cAMP slowed growth down a little bit, which is not surprising,as cAMP is a powerful metabolic signal molecule. Transfering the cells to the starvation medium MIV stopped their growth, and even caused quite a drop in cfu/ml, but after a few hours they began to grow again. This could be because A. pleuropneumoniae differs from H. influenzae in being able to synthesize its own pyrimidines - we would need to check its genome.

The lower graph shows the transformation frequencies of the cultures at the same times the cfu were measured. Cells in BHI did become quite competent when the culture density got high, just as in H. influenzae. Transfer to MIV rapidly stimulated competence, but only to the same level that develops 'spontaneously' when the culture gets dense in BHI. (In H. influenzae MIV competence is about 10-100-fold higher.) Adding cAMP to the BHI didn't appear to affect competence at all; the slightly lower competence is likely an indirect effect of the slightly slower growth rate.

This is prettier time course than the one I was trying to replicate, so this will probably be the figure that goes into the manuscript.

Praise those who post Excel Tips!

Here's a graph of some data:
How do I tell if the blue slope is significantly different from either of the grey slopes? I know just enough about statistics to know that there will be a way to test this, but not enough to do it. And the post-doc I've relied on for statistics help just moved on to a second post-doc position on the other side of the country. What to do?

Google the problem, of course. I think I searched with 'calculate confidence interval for slope, and that led me to this page, one of a collection of Excel Tips for scientists and engineers posted by one Bernard Liengme, a retired professor of chemistry and lecturer in information systems at St. Francis Xavier University in Nova Scotia.

At first the page looked quite daunting. The post-doc confirmed by email that this was instructions for doing what I wanted, but didn't offer to do it for me. I then tried clicking on the link to the sample workbook, which gave me an actual Excel file with the example calculation all set up. So I just did to my data what the example did to its, and presto, I have the confidence intervals for my lines! it's a bit embarrasing to admit that I don't know what the "INDEX(LINEST" command does, but then I don't know what's in the secret buffers and columns of the kits we use either.

So thank you Dr. Liengme! Note - a new edition of his book A Guide to Microsoft Excel 2007 for Scientists and Engineers is available in paperback from Amazon.

p.s. The 95% confidence interval for the blue line overlaps slightly the intervals for the grey lines.

Another time course to do

We convinced our A. pleuropneumoniae collaborators that the manuscript should be submitted in its current form after I do another replicate of the time course (and with some genome-analysis data presented as a table rather than just described in a sentence in the text). So now I first need to do a test of expression-time requirements for starved and growing cells, which should be relatively simple.

 

Quick plan: start with frozen starved cells, thaw cells, resuspend half in fresh starvation medium (to get rid of the glycerol they're frozen in) and half in a larger volume of rich medium.  Add DNA to the first cells, incubate 15 minutes, add DNase I, incubate 5 minutes, add an equal volume of rich medium, continue incubating.  At intervals (0, 10, 30, 60 minutes) take samples, dilute and plate on plain and kanamycin plates.  Incubate the rich-medium cells until they're moderately dense (OD about 1.0), then add DNA and DNase I and sample as above.  Total of 8 samples, needing about 32 plain plates and about 50 kanamycin plates.  This time include a no-DNA control.

Then, do another time course like the one I did last week.

Manuscript genuinely nearing completion?

One of the bioinformatics manuscripts on my desk (on the shelf, on my office floor) has been 'nearing completion' for so many years now that I'd genuinely come to see this as its permanent state.  

The work began about 10 years ago as a collaboration with a colleague in Taiwan, produced interesting results about the effects of uptake sequences on proteomes, and was partly written up and then set aside when the student who had done the bioinformatics graduated and went on to unrelated work.  At that stage I had a manuscript that was fine in some parts but flawed in others. 

About four or five years ago we began a new collaboration with bioinformaticians in Ottawa, initially on another project, but later on a new version of the old proteome project.  That produced lots of data and a new draft manuscript, but we kept finding little problems with the data and getting new ideas for analysis.  And I kept getting setting it aside to work on other things, as did my Ottawa colleague.  

But I can see the light at the end of the tunnel now, and am seriously hoping to get the damned thing submitted by Christmas.  So yesterday I sat down to try to remember where it stood, and to look at the latest (hopefully final) data.  It looks good enough, so I just need to lower my standards and get it done.  (Maybe drinking some of the beer in the lab food fridge will help.)

Time course results and prospects

Well, my execution of the time course experiment was near-flawless, but the results leave a bit to be desired.  Once again the colony counts were erratic, and some of the results are inconsistent with results of previous 'replicates'.  (Is replicate the right word here?).

I do have a new hypothesis to explain the colony-count problems: too short of an 'expression time' for the antibiotic resistance allele.  When cells recombine an allele coding for an antibiotic-resistant version of a protein, they don't instantly become resistant -- some time is needed for the new allele to be transcribed and translated into protein, and full resistance may take an hour or more.  However this matters more for some antibiotics and some forms of resistance than for others.  

With kanamycin and H. influenzae, experiments I did when I first set up my lab showed that cells could be spread on kanamycin agar plates right away (15 minutes for DNA uptake, 5 minutes for DNaseI, maybe 5 minutes for dilutions and plating), and every cell that had the new allele could form a colony.  When I started working with A. pleuropneumoniae I was told to use a very high concentration of kanamycin to prevent sensitive cells growing on the kanamycin plates, and found that cells did need an hour for expression of the allele before being able to form colonies on plates.  But I tested lower concentrations and found that sensitive cells couldn't grow at a much lower concentration.  In writing up that test I speculated that maybe they wouldn't need expression time to grow on this concentration.  But I seem to have then just gone on to do subsequent experiments without expression time without ever really testing whether it was needed.  So maybe my erratic results and low colony counts on kanamycin plates are because many cells that had acquired the resistance allele didn't have time to become phenotypically resistant before they encountered the kanamycin.

To resolve this, I can thaw out and transform some frozen competent A. pleuropneumoniae cells and test their need for expression time.  But I now wonder if these cells, having been starved to induce competence, might actually need less expression time than the cells growing in rich medium, because the antibiotic causes most of its killing when cells are actively replicating their DNA.  This would be consistent with my results, because the plating problems did mainly happen with growing cells. That's an interesting issue in its own right, and but would entail doing a more complicated test.  If the tests showed that expression time was the problem (at least a big part of the problem), I'd redo the time course.

The issue may be moot, because we may now leave the time course out of the manuscript.  One of the authors from the other research group (the leader of the group) strongly feels that we need to do more experiments before submitting this for publication, even as a 'Note' to a fairly minor journal.  But of course he wouldn't be the one doing the experiments, and the rest of us think we should just send it in and see what the reviewers think.  I and the postdoc can also think of desirable experiments, but our lists don't overlap with his.  So we'll try to persuade him by sending him our list and arguing that we can't possibly do all of these experiments, so let's wait and see which ones the reviewers might want us to do, rather than trying to read their notoriously unpredictable minds. 

Good and bad news

The good news is that my test transformation worked quite well - the transformation frequency was a bit lower than I would like, but the numbers of colonies produced from the different dilutions were just what they should have been, and the colonies themselves were both vigorous and uniform.

The bad news is that our work-study student has the flu, so I won't have any help with the big experiment. But of course it isn't really that big of an experiment, just a full day of keeping track and paying attention and neglecting other responsibilities.