Thursday, September 28, 2006

"On it's way..."

To having a reduced genome. So, it turns out Hflu is barreling headlong into a future with a reduced genome, including all the benefits: low GC content, few transcription factors, more pseudogenes, etc. And what's to stop it there? Eventually, if all goes according to Hflu's cunning plan, it will end up a small pile of adenine and thymine - forever dispensing with all those useless Gs and Cs.

This Douglas Adamsesque view of how the universal tape will play out for our favourite little bug has evolved out of a small wager in our lab. An all knowing post doc (the one who foresees Hflu eventually shedding it's too-large genome) predicted that Hflu's intergeninc regions has lower GC content than does coding sequence. An over-confident grad student who felt he had his fingers on the pulse of Hflu's intergenic regions figured that the GC content would be fairly constant between genic and intergenic regions. He reasoned that because Hflu's average GC content (38.14%) is about the same as E. coli's intergenic regions (~40%), and as E. coli is THE model organism, whatever works for E. coli should work for Hflu. This would allow Hflu to maintain a constant GC content throughout genic and intergenic regions. Because E. coi and Hflu have very similar transcriptional regulatory networks and employ many of the same transcription factors, it seems a fair assumption that they would have intergenic regions with comperable composition. However, I've just done the math (ie. used Word to count the number of A,T,G, and Cs in Hflu's 220,505bp of intergenic sequence), and it turns out that Hflu has only 33.2% GC in its intergenic regions.

So, what pressures select for low GC intergenic regions, and would E. coli go lower if it could? Melting temperature doesn't seem like a big deal; RNA polymerase melts DNA at AT rich -10 regions, but this only accounts for a small portion of intergenic sequence. More likely, DNA flexibility is an advantage. Many transcription factors bend DNA, and many repressors form DNA loops, while larger nucleoprotein complexes (which involve multiple proteins binding in close proximity) involve DNA bending and kinking. As A-T runs are more flexible than G-C runs, AT rich intergenic regions may be advantageous because they allow for DNA deformation by regulatory factors.

Tuesday, September 26, 2006

Good news Band news

Sorry, dear blog, for neglecting you for another 7 days. It's been a most productive week, which included: Some nice protein data, an unfortunate revelation about "replicating" data, an NSERC application written, and sleepless nights.

For the past few weeks, I have been having a tough time quantifying relative Sxy levels between cells in different growth conditions. I have been plagued by poor blots and poor protein stability. Here, though, repetition of experiments and long days has remedied most of the problems.

The major dissapointment and frustration came from repeating an experiment to test whether knocking out purR relieves purine nucleotide repression of transformation in Haemophilus. Background: when cells are transferred to MIV containing AMP or GMP, the cells do not transform. Two weeks ago I found that cells with mutations "sxy-1" and "sxy-2" (which result in up-regulation of the sxy gene) are less sensitive to AMP repression, but are repressed by GMP to the same extent as WT cells. Quantifying Sxy protein showed that there is lots of Sxy in sxy-1 and sxy-2 cells, so it is not limiting to DNA uptake/transformation. This suggested that another regulator, quite possibly PurR, repressed transformation in the presence of purine nucleotides.

Last week, I tested whether cells lacking purR are sensitive to GMP repression (we hypothesized that they would not be repressed). I only tested the double mutants (purR-, sxy-1/purR-, and sxy-2/purR-) for sensitivity to nucleotides, and compared the results to my earlier transformation data from WT, sxy-1, and sxy-2. At the same time, I changed the concentration of nucleotides from 1mM to 0.5mM because cell growth was inhibited by the presence of nucleotides and I hopped to aleviate this, while preserving the regulatory effect, by decreasing the nucleotide concentration. The purR- results were identical to WT, whereas the sxy-1 and sxy-2 strains were 100x more competent in GMP if they lacked purR. Because of the consistency between the WT and purR- strain, I mistakingly concluded that the two independent experiments were directly comperable.

Late last week I repeated the experiments. The results for sxy-1/purR- and sxy-2/purR- strains were identical to the previous experiment, however WT, purR-, sxy-1, and sxy-2 cells all demostrated higher transformation frequencies, effectively eliminating the purR effect I thought I had detected earlier.

The bioinformatic evidence that a competence gene, rec2, is PurR repressed is very strong. Thus, we cannot yet abandon a role for PurR in regulating competence. In the next few days I will again test whether PurR represses competence, but this time I plan to use guanosine and hypoxanthine in the hopes that they will be less toxic to cells than are GMP, and especially, AMP, and that these nucleosides will have a similar tranformation-repressing effect on cells. First, though, I must confirm that two former lab members working on nucleotide supplementation had similar resuts with nucleosides: one has reported that guanosine has a strong repressing effect.

Tuesday, September 19, 2006


A comment to my last post just coined the term "spankleen". I think this rivals "Ribowitch".

Monday, September 18, 2006

Nalgene ≠ plastic

Last week, a bold pink note was posted the side of our tub for dirty plastic dishes that declared "Nalgene ≠ plastic" and also that "bottles and flasks ≠ cylinders and beakers". This is important because we were separating plastics from glassware so as to wash the former with a mild detergent and the latter with harsh ol' Sparkleen - sorting them correctly will economize on space and expensive Nalgene cleaning products. What the somewhat cryptic note was telling us was that PC ≠ PP (ie. Polycarbonate ≠ Polypropylene).

Polycarbonate (PC) is the strongest thermoplastic (meaning it can be heated to a melting point and reshaped). It is clear, autoclavable, and can handle high centrifugal forces. PC is composed of dihydric phenols joined through carbonate linkages. The linkages are subject to chemical reactions with bases and strong acids and can suffer hydrolytic attack during autoclaving. Nalgene flasks and media bottles are made from PC - this explains why they become foggy over time and why they require gentle detergents.

Polypropylene (PP) is translucent, autoclavable and has no known solvent. This sounds great, but a drawback is that polypropylene products are brittle at 0ºC and can crack or break if dropped.

An easy way to distinguish between products made from these two plastics is to look at transparency (PC products are transparent (see-through) whereas PP products are only translucent), check the bottom of the vessel for a PC or PP label, or (the geekiest way) stick them in water: PP is lighter than water whereas PC is heavier.

Tuesday, September 12, 2006

Gels/manuscript/PurR day

After several days of working to wrap up the CRP-S manuscript, I'm looking forward to running a few more protein gels today. The first thing I want to do is directly compare how Sxy levels change between growth conditions for any given strain. Until now I have been comparing relative Sxy levels between strains at a specific growth condition, not tracking changes within a strain. This alternate approach means I can make direct measurements of Sxy levels for one strain on one gel, instead of using normalization to make comparisons between different gels. This is a last step in getting very solid numbers for Sxy levels in H. influenzae. At the same time, I will go back and test cell samples from 2 years ago (which I mentioned having probed with a different antibody in an earlier blog post).

Last week I found that in WT cells, production of Sxy is strongly inhibited by AMP and weakly inhibited by GMP. On the other hand, hypercompetence mutants produced a lot of Sxy no matter which nucleotide was present. The surprising finding was that transformation is equally strongly repressed in all strains upon addition of GMP, while AMP has some repressing effect. This has renewed our interest in PurR and its possible role in regulating competence. A former graduate student in the lab knocked out PurR, but didn't find elevated competence. I need to go over his experiments, because if he tested transformation in non-inducing conditions, PurR repression may have been lacking, but so were the necessary inducing signals. I suspect that he and the boss thought of this and that his note book holds some interesting results, so I must go have a look. It is tempting to think that rec2 (which has s very good PurR site in its promoter) is repressed when purine pools are high or when we add AMP or GMP to culture medium. Under this model, all of the other competence machinery is induced in the presence of exogenous nucleotides, but repression of rec2 keeps transformation frequency down. Real time PCR measurements of comA and rec2 in MIV+/- GMP will tell us immediately whether this is the case.

Thus, H. influenzae potentially uses two mechanisms to regulate competence according to nucleotide pools: 1. AMP represses Sxy production in WT cells, and 2. PurR represses rec2.

Saturday, September 09, 2006

A day of revisions

The past two days' work has yielded some very nice western blot results, but I haven't yet had time to sit down and thouroughly analyze the results. Today I am working on the revisions to our CRP-S manuscript (though I have spent the morning working on figure formats and resolution - fiddling with figures takes little thought and is nice with coffee).
In terms of western blot analysis of Sxy levels, I now have the satisfying result that sera from two idenpendently Sxy-infected rabbits (may they rest in peace) detect the same relative levels of Sxy in different samples. However, by switching rabbit sera I have lost an internal control (background) protein that I used for normalization, thus I can't directly compare absolute values from the two different sera. Oh well, one more set of gels should allow me to compare relative Sxy levels in all mutants and growth conditions so far.

Thursday, September 07, 2006

No background

Great success yesterday. My comparison of different rabbit sera to find one with sufficiently low background to detect minute quantities of Sxy yielded great results. One of the serum preps I had not tested back in 2004 when preparing antibodies turns out to have great signal with almost no detectable background. Today I'm redoing blots of cells grown to OD 0.2. Sxy levels are very low in WT and sxy-6 and -7 mutants at OD 0.2, so it has been hard to get satisfactory measures of Sxy levels for these strains. Now, I think we'll get accurate measures of Sxy levels for these strains. Later today I will run gels and blot the protein from last week's nucleotide supplementation experiments, thus tomorrow we will find out how/if Sxy levels vary significantly when hypercompetence mutants are treated with AMP and GMP.

Wednesday, September 06, 2006

Yesterday's blog today

Yesterday's short blog couldn't squeeze its way into my timetable, but it sounds the same as today's: Today I run more gels and westerns to measure Sxy levels in mutants and different growth conditions. Today I will also test various antibody serum preps to see if one generates less background on western blots - this is important because in some strains and growth conditions, Sxy levels are so low that they are not significantly above background.

Friday, September 01, 2006

A rushed post after another drought

Again, I've left little time to blog... but some intriguing results that shall be expanded upon in the next blog. After two days of timecourses - the first day foiled by slow growing cells - to test the effects of nucleotide supplementation in MIV on competence development in KW20 and hypercompetence mutants (sxy-1 and -2), the transformation frequency results are most surprising. AMP does not repress competence as strongly in the hypercompetence mutants as KW20, but GMP appears to have an equal effect on all strains. This suggests that the mutations have somehow lessened sxy's sensitivity to AMP pools, and moreover that it is AMP pools that change as the cell shifts from exponential growth to stationary phase. The GMP results are very interesting because, first, they show that although sxy hypercompetence mutants start with more Sxy protein when they are transferred to MIV, they do not have enough to stimulate competence, and second that the mutations specifically affect AMP but GMP repression. If sxy is a riboswitch and AMP and GMP are analogs to which it binds, we would expect the mutations to disrupt both equally... Next week the protein and RNA work should help us resolve a better model as to what's going on.