Assay for phage recombination

I think I now have everything I need to assay the ability of phage to recombine in different H. influenzae competence mutants (the mitomycin C arrived yesterday), so I've gone back and looked at the procedure I used.  (Yes, I should have done this sooner...)  The procedure is a bit more complicated than I had remembered, but maybe I can streamline it.


The procedure (from my old lab notebook):

Need: 

1. Lysates of two phage strains carrying temperature-sensitive mutations in different genes (at least 10^10 phage/ml).
2. Culture of the bacteria to be tested (at least 10^9 cells/ml), in culture or competence medium.
3. Culture of wildtype cells not lysogenic for the phage, to use for the lawns.
4. Incubators at 34°C and 40°C.
5. Melted 'top agar' (0.7 % agar medium) at ~48°C.
6. Normal agar plates at room temperature.

Recombination procedure:

1. Mix:  0.3 ml cells with about 10^9 phage of each type, both phage together and each separately.
2. Incubate for 10' at 34°C.  This gives the phage time to attach to the cells and inject their DNAs.
3. Pellet the cells, resuspend them in medium, and pellet them again.  This washes away all the phage that didn't infect the cells.
4. Resuspend the cells, diluting them 1/300 in culture medium.  
5. Set aside some of this culture (room temperature) to assay for 'infectious centres'.
6. Shake this culture for 90' at 34°C.  This time allows the phage to complete one cycle of infection.  The infected cells will burst, releasing the progeny phage.
7. Add  a few drops of chloroform to the culture to kill the remaining cells, and centrifuge the culture to remove dead cells and debris.  Transfer the supernatant ('lysate') to a fresh tube.

Assaying the results:

1. Put about 0.1 ml of the wildtype culture (about10^8 cfu) into each of a series of numbered glass culture tubes, ideally at 30-34°C.
2. Dilute the phage lysates or the set-aside culture in culture medium.
3. Add 0.1 ml diluted or full-strength lysate to each culture tube.
4. Incubate 15' at 34°C.  This allows phage to attach to cells and inject their DNA.
5. Add 5 ml top agar (2.5 ml?) and pour onto the appropriate numbered plate, rocking to quickly spread the top agar before it sets.
6. Incubate the plate at 34°C or 40°C overnight.
7. Count plaques.  
8. For the lysates, the recombination frequency is the titer of plaque-forming units at 40°C divided by the titer at 34°C.  For the set-aside culture this ratio gives the proportion of infected cells that produced recombinant page.

To get the true recombination frequency requires titering the lysates, but in the old experiment I'm looking at (#125) the frequencies of infectious centres that contained recombinants were reliable indicators of the recombination frequencies in the lysates.  For log phase cells, 2 x 10^-6 of the phage in the lysate were recombinants and 5 x 10^-4 of the infectious centres contained recombinants; for competent cells the numbers were1.9 x 10^-3 and 1.7 x 10^-2 respectively.  So maybe I can just plate the infectious centres.  I'll try both the first time and see if the correlation is reproducible.

One concern with plating the infectious centres is that recombination may occur in the wildtype cells in the lawns.  The opportunity is there because every mixed-infection cell will release a mixture of phages that may multiply infect neighbouring cells.  However these cells aren't competent so phage recombination is probably not a concern.  If the background is high I could use rec- cells for the lawns.

Clean boring results

OK, I've done the third of the experiments I planned here, and the results cleanly show that nothing interesting is going on (at least nothing interesting that warrants investigation).

I wanted to know if the peculiarities I had long-ago noticed in DNA of competent cells were reproducible.  So yesterday I grew some wildtype cells in rich medium.  Some of them I collected during exponential growth ('log phase', cell density about 2 x 10^9 cells/ml), some when the culture was approaching its final density ('stationary phase', about 10^10 cells/ml), and some I transferred to the competence-inducing starvation medium while they were in log phase.  I prepared chromosomal DNA from all three treatments, and then incubated it either in the standard DNA buffer TE (10 mM Tris pH 8, 1 mM EDTA) or in the mung bean nuclease buffer that had previously given anomalous results for competent-cell DNA.  DNA in the mung bean nuclease buffer sat at room temperature for about an hour, with and without being heated to 65 °C for 10 minutes.  Then I ran all the DNAs in a gel to check their condition.

You can see that there's no significant differences between the effects of the treatments on the different DNAs.  The competent cell DNA is in slightly shorter fragments (the biggest marker band is 29 kb), but the difference between it and the other DNAs isn't changed by the MBN buffer.  Heating the DNAs in the buffer causes the usual streaking of the bands (caused by having a lot of high-molecular-weight DNA running at the same position) to be a bit blurry, but again that applies equally to all three DNAs.

So I can put this old result out of my mind, and get to work on my planned competence and phage recombination assays.

At Northern Voice

More people than I expected, more diverse, and much more enthusiastic than the scientific meetings I usually go to.

Ancient mutants

While searching the -80 °C freezer for a missing chemical I discovered a box of H. influenzae competence mutants from 1970.  Well, I didn't freeze them in 1970, but they were originally isolated in 1970 (Castor, Postel and Goodgal, 1970 Nature 227: 515-517).  The paper gives specific information about only a few of the mutants they isolated, but the strain names go up to com-110 and a later paper refers to 70 independent isolates with abnormally low transformation frequencies.

Unfortunately, none of the strains I found (com-14, -15, -22, -23, -34, -37, -45, -47, -51 and -89) were mentioned in the 1970 paper.  If this had been a new paper, the data for all the mutants wold have been in a supplementary (online-only) table), but not 40 years ago.  So I tracked down later papers from the same group, hoping to find more information about the mutants.  I did find follow-up papers, but not much about the particular mutants I have.  One mutant (com-47) was included in a set whose DNA uptake and recombination were investigated in more detail; it takes up some DNA but not as much as wildtype.  None of these mutants were included in a group whose sensitivity to DNA damage was studied.

One of the mutants, com-51, did get a paper all to itself (Concino and Goodgal 1982  J. Bacteriol. 152:441-450).  The paper reports that the mutant cells produced normal or near-normal amounts of membrane-associated DNA-binding material when competence was induced, but this material did not remain on the cell surface.  Instead it was released into the culture medium as membrane vesicles.  The data doesn't look particularly compelling - for example, the culture medium from these cells is reported to bind H. influenzae DNA (721 cpm of 3H-DNA) but not E. coli DNA (0 cpm), but 721 cpm is only 50% above the H. influenzae DNA background (1431 cpm).  But the mutant might be useful for our work on uptake proteins and uptake specificity, even though we don't know anything about the responsible mutation (or mutations - the cells were heavily mutagenized).

In a 1981 paper the authors also characterized this mutant's phenotype and examined the cell-surface proteins it produced when competent.  Its DNA uptake was 3% of wildtype, and transformation was undetectable (<5x10^7).  The gel  analysis of iodinated surface proteins doesn't show any significant differences between wildtype and com-51.

My experiments: might I have a plan?

Classes have been over for a month and I have yet to do an experiment!  For shame.  But after going over everyone else's projects I think I have a clear idea of what I should be doing.

1. Measure starvation-induced competence for each of the competence-gene knockouts:  This will be replicating measurements done by an undergraduate over the past 6 months.  I'll just do each strain once, provided my results agree with hers.  I'll also freeze some of the competent cells.

2.  Assay phage recombination in the competent and non-competent mutant cells:  Old experiments using temperature-sensitive mutants of the H. influenzae temperate phage HP1 showed that recombination between the ts loci is more frequent in competent cells and completely dependent on the host RecA pathway (undetectable in a rec1 mutant).  Phage recombination is much more efficient in competent cells than in log-phase cells; this is true both when competence is induced by starvation of wildtype cells (in MIV medium) and when it is induced by the presence of the sxy1 hypercompetence mutation (in rich medium). 

But phage recombination is unexpectedly blocked by a rec2 mutation.  (The baroque history of rec2 is described in this post.)  The rec2 mutant also does not have the peculiar DNA aberrations described in competent cells (more below about these).  So I'm wondering if phage recombination can give us a transformation-independent window into DNA metabolism in competent cells.  Hence my plan to test all the mutants.

The recombination assays are simple in principle: just infect cells with a mixture of two ts phages (ts1 and ts3), each unable to form plaques at 40 °C.  The infections are done at the permissive temperature (34 °C), but the resulting lysates are titered at both 34 °C and 40 °C.  The ratio of plaque-forming units (pfu) gives the recombination frequency.  But in practice they're fussy.  I need to have incubators at both temperatures, which will take some arranging as we only have one incubator at present, set at 37 C.  I also need to fuss with getting the temperature control just right, as H. influenzae is pretty picky about this, and to make lysates of the mutant phages that have sufficiently high titers that I can do mixed infections (need more phage than cells).  To make these lysates I need to grow up the lysogens carrying the wildtype and mutant phages, and so I first need to find out whether they survived the big freezer meltdown of 2005.  I may also need to buy fresh mitomycin C to induce the phage - our stock has been stored in the freezer but it's more than 20 years old.

While I'm surveying competent wildtype and mutant cells I'm going to revisit an unsolved old problem - the unusual structural features of DNA in competent cells.  This was first reported by Leclecrc and Setlow in 1975, based on studies using denaturing sucrose gradients which suggested that the chromosomal DNA of competent cells had single-stranded regions, such that when denatured the average fragment length is about 200 kb (if I've correctly converted 5x10^7 Daltons to kb).  David McCarthy later used electron microscopy to examine competent cell DNA; he found single-strand gaps and 'tails' in DNA from competent cells.  The gaps were not seen in a rec2 mutant, but the tails were seen in both rec1 and rec2 mutants.  As a post-doc I tried to investigate this, using high molecular weight DNA purified from log-phase and competent wildtype cells.  I incubated the DNAs with Klenow and 32P nucleotides to tag any gaps - this labelled the DNAs of competent cells and log-phase cells identically.  DNA from stationary-phase cells was labelled less, presumably because of the absence of replication forks.  I also tried digesting the DNAs with nuclease S1, which preferentially cuts single-stranded DNA, but I had very odd results, as the competent-cell DNA migrated very strangely after incubation with the special buffer used for S1 nuclease (NaOAc ph 4.5 10 mM Zinc, 1.5 M NaCl).  It's this strangeness that I now want to revisit, so I'll just make DNA from competent and log-phase cells the usual way, incubate it with this and other buffers, and run it in gels with and without restriction digestion. 

The Research Associate's experiments, part 2

The second big component of the Research Associate's recent work is on regulation of (and existence of) competence in E. coli.  (The rest of her work is described in the previous post.)  We've shown that E. coli has homologs of all but one of the genes known to be needed for competence in H. influenzae, and these genes are induced when the competence-specific regulator Sxy is artificially induced.  BUT, we haven't been able to detect transformation, and we don't know of any conditions that naturally induce Sxy.  Because Sxy in E. coli appears to work very similarly to Sxy in H. influenzae, we're also hoping that some questions will be more easily answered in E. coli, which is a more tractable system.

Questions:  I.  About the regulation of Sxy expression in E. coli

Q. 1:  How is transcription of the E. coli sxy gene induced? 

We don't know.  Nobody knows.  See previous posts.

Q. 2:  Is the E. coli sxy gene post-transcriptionally regulated?

First, we needed to find the 5' end of the sxy transcript?  The RA has now mapped this (after some strenuous battling with experimental artefacts).  It's well upstream of the start codon (~100 nt?), as is the case in the Pasteurellaceae and in Vibrio cholerae.  So there's lots of potential for post-transcriptional regulation, as has been shown in H. influenzae and V. cholerae.

Prediction of secondary structure of the mRNA sequence using the program mfold shows that it can fold into complex secondary structure, but we'd need experimental evidence to show that this structure is biologically significant (mfold will fold just about anything).

Questions:  II.  About what E. coli Sxy protein does for E. coli

The results of the experiments described below are quite consistently negative, so the RA's strategy is to cover all the possibilities so we can at least write a solid negative-result paper.

Q. 3:  How does Sxy stimulate transcription of genes with CRP-S promoters?

CRP-S sites are variant CRP sites where transcritional activation requires both CRP and Sxy.  We have published genetic evidence that stimulation of transcription at CRP-S sites depends on direct interaction between the CRP and Sxy proteins - assays using cross-complementation between H. influenzae and E. coli induce transcription much more strongly if the CRP and Sxy proteins both come from the same species.  We also have unpublished evidence.  In vitro, far-Western blots show that CRP and Sxy physically interact, and pull-down experiments show that pulling down Sxy brings down CRP.

But Sxy doesn't bind to CRP-S sites in bandshift experiments (in vitro) whether or not CRP is present, and adding Sxy doesn't improve CRP's poor ability to bind to them.

A plan?  Construct a fusion of the candidate regulatory parts of the sxy gene (= promoter, 5' untranslated region and 5' part of the coding sequence) to a reporter gene.  Then mutagenize the sxy part and screen for changes in expression.  The mutagenesis could be done randomly with degenerate PCR oligos (???) or be site-directed.  Changes in expression would give clues about regulation, and any high-expression mutations could be tested for induction of transformability (see below).

Here's another idea.  How about we put the E. coli sxy gene, with all its candidate regulatory sequences, into a H. influenzae sxy knockout, mutagenize the regulatory region by transformation with a degenerate sxy PCR product of some sort, and then select for hypercompetence mutants as we did to get the original H. influenzae sxy mutants?  Then we can sequence the sxy genes to find mutations that increase expression (or activity).  I suppose the E. coli genes could be on a plasmid (or plasmids) rather than in the chromosome.  I can't remember whether we've shown that E. coli sxy complements a H. influenzae sxy knockout - it should, based on the E.coli experiments.

We should also just first do a time course etc. to see how replacing the Sxy gene changes induction.  For the time course we'd probably want to put the E. coli CRP gene in too, so we'd get higher induction of the CRP-S genes and thus more sensitive transformation assays.

Questions:  II.  About what the E. coli CRP-S regulon does

Q. 4.  Will expression of E. coli Sxy ever induce transformation in strain K-12?

The RA has developed an assay for chromosomal transformation in E. coli, using chromosomal DNA of a strain carrying a KanR insertion in the crp gene, or a TetR insertion in the purE gene.  Sxy is artificially induced, from either a high-copy plasmid or a low-copy plasmid.  BUT, the big problem is that we have no positive control - we don't have any conditions or genotypes where giving cells this DNA does produce transformants.  Transformation requires both that the cells take up DNA and that the DNA recombines into the chromosome.  In H. influenzae transformation is a very sensitive assay for DNA uptake because the recombination is quite efficient.  But in E. coli we have no idea whether transformation would be a usual outcome of DNA uptake.

One solution is to artificially enhance the efficiency of recombination by turning on the phage-derived recombineering genes she's been using to create mutations.  In principle this means we only need to assay for uptake.  So she's done this, inducing the recombineering genes by heat-shock (they're in the chromosome under the lambda CI857 ts promoter).  The target for recombination is the H. influenzae comM gene on a plasmid, and the recombining DNA is a PCR product (i.e. dsDNA) of a comM::SpcR fragment.  When the SpcR fragment is introduced into the cells by electroporation, she gets thousands of transformants.  The cells also contain an IPTG-inducible sxy gene, and when this is induced along with the recombineering genes, she gets a few SpcR colonies without electroporation.

Are these natural transformants?  She wants to rule out the possibility that the temperature shock used to induce the recombineering genes somehow allows some SpcR DNA to get into the cell.  She's going to do this by using a different recombineering strain, one where the phage genes are on a plasmid and induced by adding the sugar arabinose rather than by heat shock.  Another advantage of using this strain is that, if she gets what appear to be transformants, she will be able to test the effect of knocked-out competence genes by simply moving the recombineering plasmid to one of our Keio strains carrying chromosomal competence-gene knockouts.

However we still need to worry about the difference between single-stranded and double-stranded DNA coming into the cell - I think the recombineering proteins handle these very differently.  One control would be to denature the PCR product by heating it before giving it to the cells.

She's also tried inducing sxy in the  K-12 strain that Steve Finkel used to show that (1) E. coli cells can take up DNA and use it as a nutrient, and (2) this uptake needs homologs of H. influenzae competence genes.  In principle we know that this strain can take up DNA, but our visiting student last year was unable to replicate this result.  In an case, this strain did not transform with her marked chromosomal DNA.

Q. 5.  Does expressing Sxy promote recombination?

She also tested transformation by electroporation without inducing the recombineering proteins.  Whether or not Sxy was induced, she saw no transformants.  This tells us that Sxy does not activate recombination pathways.


Q. 6.  Does providing the H. influenzae pilF2 gene enable E. coli K-12 to transform when Sxy is induced?

The only H. influenzae gene needed for competence that's not in E. coli (K-12 and other sequenced strains) is pilF2.  It's in all competent Pasteurellaceae and in Vibrio cholerae.  (I'm sure I already wrote this somewhere but I can't locate it now.)  She's provided it on the sxy expression plasmid, both under its own promoter and under the IPTG-inducible promoter, but these cells didn't transform either.

Q. 7.  Will expression of E. coli Sxy ever induce transformation in non-K-12 strains?

She's tested many strains of the ECOR collection (a reference set of diverse E. coli strains isolated from many different locales and hosts) for transformation during growth and stationary phase in the rich medium LB, using her chromosomal DNA (probably crp::KanR).  No transformants were seen.  She also tested the cultures for expression of the Sxy-induced pilin protein; none was produced.  With the help of a summer volunteer undergrad she's now going to put an inducible sxy plasmid in these strains and retest them.

Q. 7.  Does expression of E. coli sxy and the CRP-S regulon provide E. coli with benefits other than competence?

The RA hasn't been working on this specifically, but we need to think about it if her results continue to be negative.  If it's true that E. coli cannot take up DNA by the natural competence mechanism, why does it have intact versions of all the necessary genes except pilF2, under a Sxy-controlled CRP-S regulon?  If these genes were just leftovers from a competent ancestor, we would expect to see some loss-of-function mutations in them, if not in K-12 then in some of the other sequenced strains.  (Has anyone checked the alleles in the other sequenced strains?)

How carefully have we checked the phenotype of the E. coli sxy knockout?  Might it be defective at anything?  In the RA's E. coli sxy paper (2009) we compared long-term survival of wildtype and sxy-knockout cells in LB.  The mutant reached the same initial cell density as the wildtype, and it survived long-term culture just as well.  But it did not compete equally with wildtype when the two strains were cultured together, a phenotype that Steve Finkel's work suggested was due to inability to sue DNA as a nutrient.

The Research Associate's experiments, part I

While I've been paying attention to other things (teaching, writing, proposing), the Research Associate has been doing a lot of work on the projects we're funded to do.  Below I'll highlight in purple the experiments that I might take on.

Recombineering:  The goal was/is to create marked (selectable for antibiotic resistance cassette) and unmarked deletions of all the H. influenzae competence genes.  This has required an enormous amount of work, much more than was originally expected.  The last step, converting the marked mutants to unmarked, turned out to be particularly troublesome because the counter-selection doesn't work in the standard lab strain.

But I think we now have marked knockouts of every gene in the competence regulon, but unmarked mutants for only some.  These are on the back burner for now, though she's still creating knockouts of comI and comJ (implicated in competence though not regulated by it) and mutS (for study of recombination).  She also knocked out the sxy gene of a related species, Actinobacillus suis, for a collaborator.  One project for me is to follow up on the characterization of these competence mutants (begun by an undergrad), rechecking their starvation-induced transformation frequencies.

Competence differences in H. influenzae strain 86-028NP ('NP'):  This is the low-competence strain the postdoc is using for his sequencing of recombination tracts and the planned mapping of genes responsible for competence differences.  The RA noticed that, although this strain has homologs of all the genes known to be needed by H. influenzae strain Rd for transformation, it lacks several competence-induced genes with unknown functions.  So she's putting the Rd genes into the NP chromosome to see if this changes their competence.  This should be easy as she already has the Rd genes cloned in the recombineering vector, and the counter-selection that fails in Rd works fine in NP.


Regulation of competence by purines and purine nucleotides:

The big project that's most important is understanding how purines affect competence in H. influenzae.  The new work on this project has two fronts, repression of CRP-S genes by PurR and post-transcriptional control of Sxy expression by purine nucleotides.

Front 1, regulation by PurR:  The lesser front is finding out whether the purine repressor PurR affects transcription of any competence genes.  When one of the purines guanine and hypoxanthine is available, they bind to PurR and enable it to bind its recognition sequence.  This sequence is present in the promoters of most genes involved in de novo synthesis of purines.  I initially thought it was also present in the promoters of several CRP-S genes but the RA and others have analyzed this more rigorously and find that the only strong candidate is in the rec2 promoter.

Our previous work on the role of PurR in competence has been compromised by problems with what we thought was a good PurR knockout strain.  First we found that strains had been mixed up, and ten that the knockout didn't remove the part of the gene that codes for the DNA-binding domain of PurR.  Now the RA has made a clean mutation that removes the whole gene.  She's also knocked out the purH gene; this gene codes for the last step in purine synthesis, and the knockout allows us to disassociate competence effects due to PurR regulation from effects due to synthesis of purines.  She's put the two knockouts together and also combined them with some sxy-hypercompetence mutations (see below).

One simple way to test for PurR regulation of rec2 is using some fusions to the E. coli lacZ gene, made years ago by Michele Gwinn.  We have her fusions to two CRP-S genes, comA and rec2 - comA makes a good control as it doesn't have a candidate PurR site.  The RA has made the purR- derivatives of these fusion strains and tested them in rich medium, using cells in log phase, at the onset of slowed growth, and after overnight culture.  None of the conditions showed much difference in expression of the fusions with or without PurR.

...pause while I diagram what we should expect...  First, we expect PurR to be active (repressing) throughout growth in sBHI, because our microarrays showed no induction of PurR-repressed purine genes.   We also expect little or no expression of comA, rec2, and the other competence genes (CRP-S genes) in log phase, because sxy is not transcribed much or translated efficiently.  When growth slows we expect low-level expression of the CRP-S genes.  If PurR represses rec2, we expect to see higher beta-galactosidase expression in the purR- rec2 fusion strain than in its purR+ control.  But she didn't see that. 

This might mean that PurR doesn't regulate transcription of rec2.  But the expression levels of the fusions were very low (mostly less than 40 Miller units, whereas starvation-induced cells produce several thousand units).  It would be good to repeat these experiments after adding cAMP to all the cultures to give higher expression of the CRP-S genes; if we don't see any effect of PurR, probably there isn't much effect.  She's now also done careful measurements of the baseline expression of these fusions, data we need to interpret the results of whatever manipulations we now do.  I don't know if we've ever monitored the effects of cAMP on expression of these fusions in rich medium - it might be easier to just do it now than to find out whether we've already done it...

The presence of a functional purine-biosynthesis pathway in these cells shouldn't have mattered, but the purR- cells did differ from the controls in this, so it would be good to repeat the experiments with the purH knockout added to all strains. 

Another way to test for PurR repression of rec2 would be to put the H. influenzae lacZ fusions into E. coli, where it will be easier to test the effect of PurR and purines (because we can grow cells with and without various purines.  Unfortunately we don't have plasmid clones of the fusions, just  H. influenzae chromosomal fusions.  The RA thinks it might be easiest to just remake the E. coli fusions rather than cloning them from the H. influenzae chromosome, but I think that at least one of the fusions is a simple integration of an E. coli plasmid.  These cells should contain a low frequency of excised plasmid - if so, simple transformation of E. coli with a cell lysate might give the desired plasmid clone.

Another test would be direct assay of bandshifting.  The RA wondered if we might be able to get purified PurR protein (E. coli) from someone.  But I just looked at the old E. coli PurR paper (1988); they did bandshift assays using crude cell extracts, so maybe we could do that too. 

We also want to know how widespread competence regulation by purines is in other Pasteurellaceae.  The RA has found that the putative PurR binding site is also present in the rec2 promoters of other Pasteurellaceae, and E. coli.

Front 2:  Regulation of sxy expression: 

The RA has confirmed and extended our previous work showing that purine nucleotides (AMP and GMP) prevent competence development in starvation medium.  Most of that work used quite high nucleotide concentrations (5 mM), and some of our experiments found that these were high enough to inhibit cell growth.  So she's meticulously repeated these using concentrations of 1.0 and 0.1 mM AMP (3 replicates).  1.0 mM was enough to reduce transformation by 500-fold, and 0.1 mM by 40-fold.  But the 1.0 mM AMP also inhibited cell growth - cells usually double once after transfer to the starvation medium, but their numbers didn't significantly increase in 1.0 mM AMP.  Cells transferred to starvation medium with 0.1 mM AMP doubled normally.  This is excellent, as it gives effects on competence at concentrations that are unlikely to cause other problems.  But I'd still like to have some better controls, to confirm that the competence effects we see are really directly due to specific effects on competence genes, not to more widespread effects.  One possibility is to use a CRP-N-regulated gene (not responsive to Sxy) as a control.

She's done similar experiments in two other competent Pasteurellaceae, Actinobacilus pleuropneumoniae and A. suis.  Our collaborators have found strains of these species that transform at sufficiently high frequencies that the effects of added purines could be tested, and the RA has shown that AMP reduces their competence in the same way as in H. influenzae.

She also tested the new purR knockout (in H. influenzae).  Its transformation frequency in starvation medium is down 5-fold.  This may be because the cells are constitutively synthesizing their own purines and so are less 'shocked' by the sudden absence of purines from their surroundings.  They were also less sensitive to addition of AMP;1.0 mM AMP reduced transformation only 10-fold, and 0.1 mM had no effect.  This reinforces the hypothesis that sudden purine depletion contributes to competence induction, and that enrichment of the starvation medium with AMP decreases competence directly and not by some general perturbation of metabolism or gene expression.

These effects were reduced when the cells carried sxy-hypercompetence mutations that increase the efficiency of translation of sxy mRNA.  1.0 mM AMP reduced transformation of a sxy1 mutant only 20-fold, and 0.1 mM no more than 2-fold.  She's done 3 replicates so this is a solid result.  She also tested a couple of our other hypercompetence mutations; in both of these she only tried 0.1 mM, and saw no effect on transformation.  This is consistent with our hypothesis that AMP acts by preventing translation of sxy mRNA.  She has saved cell pellets of all the competent-cell preps she used, so these can be assayed for expression of Sxy.  An excellent control would be to assay the pellets for presence of some other protein that's induced by the starvation regime but not regulated by Sxy.  Again, one of the cAMP-induced CRP-N-regulated proteins would be good, but we'd have to make our own antibody first (either purifying the protein or specifying a synthetic peptide).

She's also modified our antibody-based assay (Western blot) for Sxy.  The grad student who developed it used a detection method that was very sensitive, but his blots had very high background.  She's found that, with a less-sensitive assay, the Sxy bands are still very clear but the background is greatly reduced.  It's not entirely eliminated, which is a good thing because the few background bands serve as excellent controls for normalizing the band intensity (provided, of course, that their proteins aren't altered by the starvation regime).


E. coli competence:

This is the second big research problem the RA has been working on.  I'll leave this for a later post.

The postdoc's manuscripts

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. 

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.

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.

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 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.

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.

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.:

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?)

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!)

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.

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.

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...

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.