duckfeet

Testing whether the CRP-S sequence confers Sxy-dependence.

2008-03-18T13:24:00.000-07:00

I am currently studying how CRP binds to and activates transcription from promoters with CRP-S sites. These promoters are unique in H. influenzae for two reasons: 1. They depend on the Sxy protein for transcription, and 2. The CRP-binding sites (called “CRP-S”) in these promoters differ at the sequence level from other CRP sites (called “CRP-N”) in the genome.

I previously tested what happens if I mutate a CRP-S site to resemble a CRP-N site. The mutations are deleterious to the promoter but allow for some transcription activation in the absence of Sxy. This result is currently in a manuscript in preparation, but the manuscript suffers from not having the reciprocal experiment of converting a CRP-N (Sxy-independent) site to resemble a CRP-S site. I attempted this experiment twice previously, first by mutating the H. influenzae mglB promoter and then by mutating the E. coli lacZ promoter; both of these promoters have CRP-N sites.

mglB presented all sorts of cloning problems until I discovered that cloning even just the 1st 10 amino acids of the gene was highly toxic to cells – ie. I could not recover plasmids that had not undergone large rearrangements. I did eventually clone and mutate the promoter, but never used it because I couldn’t conduct real time PCR to measure transcription. However, it may be worth going back and cloning the mglB promoter adjacent to lacZ and use beta-galactosidase activity to measure promoter activity.

Next I mutated the CRP site in the lacZ promoter; this was easy because our standard H. influenzae cloning vector, pSU20, already carries the lacZ promoter and lacZa gene. However, it turns out that lacZa is constitutively transcribed at very high levels in H. influenzae, even in a crp- background. Thus, the lacZ promoter is completely CRP-independent in H. influenzae and is useless for my experiments. I then cloned the lacZ promoter mutant in E. coli, but because Sxy induction experiments aren’t very clean in E. coli, the data was never very compelling. Nonetheless, the data did suggest that giving lacZ a CRP-S site reduced its stimulation by CRP, as expected, but transcription was unaffected by the presence/absence of Sxy.

The lacZ promoter mutant may be useful in future when we better fine-tune Sxy activity in E. coli, but in the meantime I really want to answer the question of what happens when a CRP-N site is converted to a CRP-S site in H. influenzae? Today I am planning the steps involved in cloning and mutating the ansB promoter/gene, which has a good CRP-N site and is strongly induced in MIV even in sxy- cells. I will also consider using beta-galactosidase activity instead of real time PCR to measure promoter activity.

Why CRP-S sites are special

2008-01-12T19:10:00.000-08:00

I’m trying to improve my ability to verbally express the significance of our CRP-S work. Here I outline a discussion I had with a former supervisory committee member who asked something along the lines of “Aren’t CRP-S sites simply low affinity CRP sites?” He asked this because many bacterial promoters use regulatory mechanisms that require a transcription factor to bind first to high affinity DNA sites and then, once the good sites are all full, less favourable DNA sites will begin to be occupied.

A classic example is the OmpR system (Figure 1). When only a few OmpR molecules are active in DNA binding (OmpR~P), the high-affinity site in the ompF promoter is bound and ompF is is transcribed. When more OmpR molecules are phosphorylated, low affinity binding sites become occupied, which turns off the ompF promoter and turns on the ompC promoter.

Below I describe the CRP-S model as it is coming to light in H. influenzae. This is a simple model that will improve as we gain a better understanding of Sxy and CRP-S function in E. coli.

Key point: “CRP-S” sites are low-affinity but highly specific CRP sites. CRP binding to CRP-S sites is mechanistically different than for binding to canonical low-affinity sites. Canonical low-affinity sites are low-affinity because CRP has a low-specificity for them, and in general protein affinity and specificity go hand in hand.

Our discovery: CRP sites in E. coli, H. influenzae, and very likely many bacteria, fall into two sub-populations: canonical “CRP-N” sites and unusual “CRP-S” sites. CRP-N and CRP-S are names that Rosie and I coined. Previously studied CRP sites in E. coli belong to the CRP-N group, for example E. coli’s lacZ promoter has a CRP-N site.

Background: E. coli CRP binds as a homodimer, specifically to symmetrical 22 bp DNA sites with the consensus half site 5’-A1A2A3T4G5T6G7A8T9C10T11. The protein makes direct contact with base pairs G:C5, G:C7, and A:T8 in the highly conserved core motif T4G5T6G7A8, and binding induces a localized kink of 43º between positions 6 and 7, wrapping the DNA around CRP and strengthening the association. Though base pair T:A6 is not directly contacted by CRP, it is recognized indirectly because kink formation strongly favours T:A6 over other base pairs. For example, replacement of T:A6 in a consensus CRP site with C:G6 causes an 80-fold reduction in CRP affinity by increasing the free energy required to bend the DNA.

Despite the extensive variation among CRP sites, until our work no significance had been attached to which positions vary. Instead, the degree of similarity of any CRP site to the consensus was proposed to generate an adaptive hierarchy that allows genes with better sites to be preferentially activated at low cAMP concentrations. Similar hierarchies must exist for all DNA-binding proteins – ie. good sites are preferentially bound at low protein concentrations. Many regulatory mechanisms exploit this hierarchy, such as OmpR’s ability to act both as an activator and a repressor. When a sufficient number of OmpR molecules are active in DNA binding, both high- and low-affinity OmpR sites are occupied; the amount of occupancy has a profound influence on the activities of the ompF and ompC promoters.

The CRP-S story is different: CRP-N and CRP-S sites are each best described by a different consensus sequence: CRP-N sites have the core sequence TGTGA whereas CRP-S have TGCGA. DNA sequences in one population differ significantly (in a statistical sense) from sequences in the other population – and we can even detect this by eye.

I have found that CRP-S sites are low-affinity binding sites for CRP in vitro, but in a special way unlike low-affinity CRP-N sites. Low-affinity CRP-N sites have multiple non-consensus bases at key positions; these prevent specific contacts from forming between the protein and DNA site. CRP-S sites are different because in H. influenzae they have all the correct bases required for binding by CRP, except that the non-consensus bases at positions 6 and 17 (see “Background” above) prevent the protein from forming a stable interaction. In other words, all of the more than ten specific contacts between amino acids in CRP and bases and phosphates in a CRP-S site can form, but the DNA site won’t kink because of bases C6 and G17.

Thus, CRP-S are a special, mechanistically distinct, type of low-affinity site: CRP has high specificity for these sites, but critical base substitutions make them poor binding sites. Consequently, CRP-S sites don’t properly fit in the classic hierarchy of high- to low-affinity CRP sites. Instead, CRP-S sites appear to occupy an alternate “binding site landscape” that is accessible to CRP only when Sxy is present. Figure 2 plots a generic CRP-S site relative to other CRP sites (not real data) and shows that when Sxy is present, CRP-S sites will be bound by CRP even when CRP levels are low. We know this to be true because competence promoters can be turned on even when there is little active CRP in the cell – such as what we see in the sxy-1 hypercompetent mutant during exponential growth. The pil-N promoter (highlighted red) is a CRP-S site that has been mutated to have the canonical bases T6 and A17; it is a high affinity CRP site.

Our working model is that Sxy in required for CRP to form stable interactions at CRP-S sites. However, I suspect that Sxy doesn’t operate through a simple recruitment mechanism. If Sxy were to recruit CRP to CRP-S sites, we would expect a strong Sxy-binding site and a weak motif at the CRP-S site. However, CRP-S sites are the only apparent biding sites in their promoters. Also, the high specificity of CRP for CRP-S sites predicts that CRP-S sites will be occupied even at low CRP concentrations (providing Sxy is present to help with DNA kinking).

Other distinguishing features: The C6 and G17 bases in CRP-S sites are important for promoter function. Changing them to canonical bases T6 and A17 makes a weaker promoter. This is opposite to other CRP sites, where changing bases to match the consensus yields a stronger promoter. This finding suggests that when CRP is able to bind alone to a mutated CRP-S site, it is not as good a transcriptional activator as a CRP-Sxy complex bound to a wildtype CRP-S site.

Conclusion: It is possible that my models and interpretations of experimental data are wrong. Instead, CRP-S sites may have C6 and G17 to make them binding sites for Sxy as well as for CRP. If this alternate interpretation is true, ie. that CRP-S sites are targeted independently by two separate proteins, it is still completely novel and distinguishes CRP-S sites from all the other CRP sites.

The complexities of gene regulation in E. coli

2007-12-28T11:52:00.001-08:00

We are currently identifying genes that belong to the Sxy regulon in E. coli. The only other well-characterized Sxy regulon was identified by our work in H. influenzae (link). E. coli’s genome is over 2-times larger than H. influenzae’s and, not surprisingly, the E. coli Sxy regulon contains more genes. The E. coli regulon has an additional level of complexity because many of the Sxy-regulated genes are likely to have additional protein regulators (ie. Sxy-regulated genes also belong to other regulons).

This additional complexity is a consequence of lifestyle. E. coli is a more versatile organism than H. influenzae: it can make most of its organic molecules from scratch (aka. from simple sugars plus a few inorganic nutrients) and it can survive in various different environments.

Because a bacterium must at all times satisfy multiple metabolic requirements, it needs to continuously balance its internal functions while exploiting a potentially ever-changing external environment. Bacteria that inhabit very stable niches (such as H. influenzae, which lives in the cavities of a human host) have a small number of transcription factors, whereas bacteria in more complex environments employ a much larger number of regulators. This relationship has been shown to scale as a power-law in which the number of transcription factors doubles twice as fast as does the total number of genes in a genome (van Nimwegen,E., 2003, Trends Genet., 19, 479), indicating that large bacterial genomes employ disproportionately more complex regulatory networks.

Thus, E. coli’s lifestyle necessitates more sensory and response systems than does that of H. influenzae. Consequently, the genes in E. coli’s Sxy regulon are much more likely to belong to multiple (possibly non-overlapping) regulatory networks in order to fine-tune their expression. This unfortunately makes studying the E. coli Sxy regulon more complicated; we can’t be confident that overexpression of the Sxy protein results in induction of all Sxy-regulated genes.

For H. influenzae, gene expression data coupled with bioinformatic analysis revealed that most genes in the Sxy regulon require only CRP and Sxy for transcription activation in standard culture conditions. In E. coli, some Sxy regulon genes will likely be repressed during growth in standard culture conditions, regardless of whether CRP and Sxy are trying to turn them on. Perhaps conducting E. coli gene induction studies in minimal medium will reduce the activities of repressor proteins and so improve our ability to detect some members of the Sxy regulon.

Fortunately, a substantial body of knowledge surrounds the regulation of some of the genes in E. coli’s Sxy regulon. Thus, although we will have trouble identifying genes that can’t be induced by Sxy in standard lab conditions, we will at least be able to integrate our Sxy regulon data with other regulatory networks.

Latest ICAP results

2007-12-10T20:34:00.000-08:00

I have now quantified EcCRP and HiCRP affinity for ICAP and four designer variants, and am currently replicating the experiments. The data looks good and supports my hypotheses, but some additional interesting features of CRP-DNA interactions have revealed themselves.

For example, CRP binding causes DNA to assume a very sharp bend of around 90º, which is achieved through two major kinks near the centre of the DNA site (each ~40º) and lesser kinks at each edge of the binding site (each ~10º). Two of the ICAP variants were designed (in part) to address the importance of the small secondary kinks for HiCRP affinity. As I predicted, HiCRP appears to need favourable interactions with a longer stretch DNA than does EcCRP, possibly to stabilize kinking, but what I didn’t expect is that I can readily detect variations in the degree to which DNA is bent by CRP. The second surprise is that when multiple CRP molecules bind to bait DNA at high protein concentrations in bandshift reactions, HiCRP appears to bind in a stepwise fashion: first one protein and then two (possibly in a cooperative manner). EcCRP, on the other hand, goes very quickly from having only one protein bound to a stage were more than two proteins bind to the same piece of DNA, seemingly in a more haphazard fashion. I suspect this is consistent with my model in which HiCRP is highly selective for DNA sites, whereas EcCRP is much less choosy and will bind all sorts of less favourable sites when the good ones are saturated.

These are interesting results, but they require much more thinking before I make good sense of them. Also, they beg for more experiments and my plate is looking pretty full considering the number of different DNA species (ie. different natural promoters) I still want to test in "simple" affinity experiments.

Success with equations

2007-11-30T00:05:00.000-08:00

I have solved the issue of excess versus limiting DNA in bandshift reactions. It turns out that my confusion arose because of the two distinctly different ways that the equation for deriving equilibrium binding constant can be derived: the common (overly simplified) equation for calculating Kd (the dissociation constant, which reflects a protein’s affinity for a DNA site) ignores the important qualifiers of the protein and DNA concentrations in the reaction. Unfortunately, the Kd equation cannot be easily illustrated in this blogger post due to my inability to write equations in a blog, so I will add the equation (and its derivatives) tomorrow when I have time to draw them out and edit this post.

The bottom line is that all of the bandshift experiments I have conducted have been informative, and now I can confidently proceed to the next step of measuring accurate Kd values for CRP binding to different CRP sites. Also, tomorrow we will test a second prep of His-tagged Sxy to see if once again CRP has been co-purified along with Sxy. If positive (ie. CRP was pulled down with His-Sxy), we can easily test how much salt needs to be added to His-Sxy to prevent the co-purification of CRP; the amount of salt needed to block the interaction will give us a sense of the strength of affinity between the two proteins.

ICAP bandshifts going well

2007-11-22T22:14:00.000-08:00

I am using the perfect CRP binding site, ICAP, to measure both the amount of protein active in DNA binding in my protein preps and to calculate EcCRP and HiCRP’s affinities for the perfect binding site and derivatives of this site. My first affinity measurements (which are expressed as equilibrium binding constants, Kobs) indicated that I had lots of active CRP molecules, but EcCRP’s affinity for ICAP was ~100-fold less than that observed by the researchers who first developed ICAP. I am the first to work with HiCRP, so there are no precedents with which to compare HiCRP binding constants.

Initially I thought that I was skewing my Kobs measurements by using too much bait DNA in binding reactions, so I started experimenting with lower bait DNA concentrations. Changing bait DNA concentrations had no effect on Kobs measurements, which was heartening in that I have nice replicate measurements and confirms that my binding reactions are resistant to perturbations. However, this didn’t explain my low Kobs measurements.

Binding reactions are set up such that CRP is presented with a great excess of non-specific DNA; for this I use poly-dIdC, an unnatural DNA molecule that doesn’t have any CRP binding sites. Using non-specific competitor DNA ensures that non-specific DNA binding by CRP won’t contribute to the bandshifts that I am using to measure protein-DNA affinity. This is important because DNA binding proteins are attracted to DNA and so spend a lot of time interacting non-specifically with DNA; when a protein finds a specific binding site, more bonds are formed between it and the DNA so the interaction persists for a longer period. I like to think that including a great excess of non-specific DNA in bandshift reactions is the most biologically relevant approach to studying protein-DNA interactions because in a cell, the vast majority of the chromosome does not have a CRP binding site. Further, I have read and have been told that affinity constants can only be reliably measured in the presence of excess non-specific DNA.

Thus, I was surprised to discover this week as I was re-reading some ICAP papers that the ICAP gang was/is using CRP in excess over ICAP bait DNA, without any non-specific competitor! My first step was to repeat my ICAP bandshift yesterday with low CRP concentrations, but with even lower DNA concentrations (and no competitor DNA). The result is very clear: in the absence of cold competitor, the Kobs value increases ~100-fold to a value similar to previously published values. Thus, in the next few days I will delve deeper into understanding the calculation of affinity constants and will revisit those wise biochemists in the Biochem department.

No matter which approach I take with my bandshifts, I am very pleased with the quality of the data and I’m only a week away from measuring all the ICAP variants. The data will be very informative and will make a great figure for the manuscript that I think is improving by leaps and bounds.

More results from Sunita and Andrew

2007-11-11T13:17:00.000-08:00

Our results that suggest Sxy binds to DNA are exciting but suspicious; all Sxy-DNA binding data can be explained by the presence of contaminating CRP in the Sxy protein preps. This is because the Sxy-DNA binding data is identical to CRP-DNA data. First, EcSxy binds DNA but HiSxy does not. Second, EcSxy greatly prefers the pilA-N (CRP-N mutant) site over the wildtype pilA CRP-S site. Third, when EcSxy and EcCRP are mixed together, only one protein binds to a DNA molecule, suggesting that both proteins target the same site (this is consistent with the pilA-N data).

The next set of experiments is clear: 1) Test whether EcSxy can bind DNA in the absence of cAMP (EcCRP cannot), 2) Test whether EcSxy binds to a pilA promoter that lacks its CRP site, 3) Use western blots to probe for EcCRP in the Sxy preps, and 4) Test DNA binding by EcSxy that has been isolated from a crp- expression strain.

However, several arguments can still be made that EcSxy does in fact bind DNA. First, EcSxy and HiSxy were isolated form the same E. coli strain using the same procedure, thus we would expect EcCRP to contaminate the HiSxy preps as well (which clearly has not happened because HiSxy preps don’t bind DNA). Second, far-western analysis has not detected EcCRP in the Sxy preps.

If tomorrow’s experiments show that EcSxy binds DNA in the absence of cAMP, two new hypotheses need to be addressed: 1) Does EcSxy prefer the pilA-N promoter not because it binds the CRP-N site, but because the CRP-N site makes DNA more bendable than the wildtype CRP-S promoter? 2) Does HiSxy fail to bind DNA because bandshift reaction conditions are not favourable for H. influenzae proteins?

Hot stuff (aka. sticky Sxy)

2007-11-08T12:39:00.000-08:00

After a grueling few days of bandshift experiments to study CRP and Sxy binding to the pilA promoter, we have made the excellent discovery that Sxy binds to DNA. Surprisingly, it appears that Sxy may bind to the CRP site in this promoter. We have several promoter constructs that lack various parts of the wildtype promoter - we can use these to directly test whether CRP and Sxy bind to the same site. We are taking a break from bandshifts this afternoon to analyze the data and plan our next set of experiments. We will begin to frame these results as a figure for our DNA-binding manuscript.

4/4 is even better

2007-10-31T17:41:00.000-07:00

Today I received the sequencing results from my second putative clone of the ICAP-9G,14C variant and it was good! Thus, I now have all of my ICAP variants and so can proceed with more bandshift analysis, while at the same time conducting CRP-Sxy bandshifts along with Sunita.

Further analysis of the first ICAP-9G,14C clone revealed that it lacked a restriction site where the sequencing primer was targeted to bind, consistent with the failed sequencing reaction that I described in my last post. That plasmid is now in the trash and I can file the experience away as another one of those funny little cloning complications.

3 out of 4 ain’t bad

2007-10-28T20:46:00.000-07:00

(It’s better than Meat Loaf’s 2 out of 3.) This week I attempted to clone 4 modified versions of the ICAP CRP-binding site. In a previous post I described my attempt to use site-directed mutagenesis to change ICAP. That approach failed, so I returned to the method used to originally clone ICAP: complementary synthetic oligonucleotides containing the ICAP sequence (or variant sequences) were allowed to anneal and the resulting dsDNA 28 bp molecules were cloned.

As described in the earlier blog post, the ICAP palindrome poses a serious problem because of the formation of very strong hairpins, which directly competes with inter-molecular annealing. Consequently, cloning synthetic oligos has a very low efficiency (I was lucky with ICAP; the single positive clone had the ICAP sequence!), and this is why I had opted to try site-directed mutagenesis. This time around, oligos were melted at 95º and then given 10 hrs to cool to room temperature, an approach that should hold oligos at high temperatures where inter-molecular bonds are stable and hairpins are not.

This round of cloning was slightly more successful. Each ICAP variant had at least 5 positive clones (ie. white colonies in blue/white screening on X-gal) except for ICAP-9G,14C (2 white colonies), which has the strongest hairpin structure of all ICAP variants.

My sequencing results came in on Friday and all but ICAP-9G,20C came back positive as having the desired sequences. Unfortunately, there was no sequencing read for ICAP-9G,20C, thus I may in fact have the desired clone, I just don’t know it. I will talk to NAPS on Monday about what may have gone wrong. The strong ICAP secondary structure had been a problem in earlier sequencing reactions, but this appears to have been circumvented by addition of betaine to sequencing reactions. Even if the ICAP-9G,20C secondary structure remains problematic during sequencing, the reaction should have at least yielded sequence up to the hairpin; the “no sequence read” is a mystery, so I really hope the folks at NAPS have a good idea of what to try next. This weekend I extracted plasmid from the other ICAP-9G,20C positive clone, so I’ll take that in for sequencing on Monday as well. Given the success of the other three clones, I remain confident that ICAP-9G,20C will also turn out well.

Covariance in CRP sites

2007-10-23T19:01:00.000-07:00

Yesterday in lab meeting I described how I will introduce base changes at various positions in ICAP to test which positions are most important for HiCRP binding (see previous blog). One important feature of CRP sites, both in E. coli and H. influenzae, is that they have A/T rich runs at either end (positions 1-3 and 20-22; see figure showing a sequence logo from alignment of 45 H. influenzae CRP sites). These A/T rich regions allow a small degree of bending in the DNA, which strengthens protein-DNA interactions. EcCRP does not directly “read” the base sequence at positions 1-3 and 20-22, instead, contacts between the protein and the phosphate backbone of the DNA are favoured when these positions are A/T rich because A/T runs are more flexible. Thus, there has been no reason to think that it matters whether there is a T or an A at position #1 (for example); either base will do. However, Heather made the great point in lab meeting that covariance between adjacent bases at positions 1-3 and 20-22 may be important and would not be apparent in a sequence logo. In other words, if position #1 is a T, it may be much better for CRP binding if position #2 is also a T; conversely, if #1 is an A, position #2 is more likely to be A.

I used MatrixPlot (which has been previously used in our lab for USS analysis, see here) to detect covariance in the 45 experimentally determined H. influenzae CRP sites. Absolutely no covariance was detected between any positions over the 22bp length. I think 45 sites should be adequate for detecting significant covariance. This result is good news because it means that when I make a point mutation in ICAP, I probably don’t have to worry that this same mutation would have a dramatically different effect on CRP affinity if it were introduced into a different site (as I had speculated before). In other words, base contributions to CRP binding may be additive and not cumulative, which makes experiments more straightforward and easier to interpret.

Mutating the ICAP binding site

2007-10-15T23:08:00.000-07:00

In my last post I reasoned that I should introduce mutations into the perfect CRP binding site, called ICAP, to measure HiCRP’s preference for certain bases at certain positions within the site. This past week I did just that (I hope). However, the mutagenesis procedure presented some technical difficulties, prompting me to experiment with a new approach.

The big problem is that the ICAP site is a perfect palindrome (see the DNA sequence in the figure; the vertical dashed line shows the axis of symmetry) and the vector sequence on either side of the ICAP clone is also palindromic, resulting in a palindrome >40bp long. The problem arises because mutagenic primers are normally designed to be perfectly complementary and extend by >20 bases on either side of the mutation being introduced. I want mutations near the axis of symmetry, resulting in primers that in most cases are themselves perfect palindromes (purple primers in figure, labeled as “old method”; not to scale). Thus, it is highly favourable for primers to form hairpins and become double stranded along their entire length; these intra-molecular bonds form more readily than do inter-molecular bonds with template DNA.

Using pcr, the mutagenic primers generate full-length linear amplicons from plasmid DNA. Complementarity between the primers allows the newly synthesized sequences to anneal as double stranded circular molecules with staggered nicks, which are repaired by the host bacterium after transformation. I reasoned that the complementarity is key, but the mutagenic primers do not have to overlap for their full length. Thus I designed primers (green in the figure; not to scale) that have a region of complementarity with one another, but only one primer has the mutation and the other does not overlap this region. This allows me to design primers that contain short palindromes, thus greatly increasing their preference for annealing to template DNA and reducing the strength of intra-molecular pairing.

I was pleased to later discover that a biotech company now markets a mutagenesis system that uses the same approach of staggering primers, giving me confidence that this approach would work. I got the satisfying result today: my positive control of introducing a stop codon in pSU20’s lacZalpha gene had a 75% success rate (ie. 75% of colonies were white when plated on Xgal). This is lower than the >95% success rate I enjoyed with the old method (which used mutations in pUC18’s lacZalpha as a positive control), but all I care is that I circumvented the primer hairpin problem and got mutants. Unfortunately, my positive control mutagenesis is not exactly like my ICAP mutagenesis, so I will need to wait for sequencing results later this week to know if the experiment really worked.

Should I make a bad site better or a wonderful site worse?

2007-10-09T11:24:00.000-07:00

In order to understand competence gene regulation in H. influenzae, we have been using the huge body of knowledge surrounding E. coli CRP to inform us about how the ortholog in H. influenzae functions as a transcription factor. Recently, bandshift analysis of H. influenzea CRP (HiCRP) binding to DNA has confirmed that both HiCRP and E. coli CRP (EcCRP) have the same perfect binding site (called ICAP, see an earlier blog about ICAP). However, I also discovered that HiCRP is much more selective about which sites it will bind; for example, HiCRP will not bind to E. coli’s lacZYA promoter, even though EcCRP has very high affinity for this site.

Eleven amino acids in EcCRP’s helix-turn-helix DNA-binding domain make direct contact with bases and/or phosphates in the ICAP DNA site; EcCRP makes fewer direct contacts with the lacZYA CRP site because some of the base pairs in this site are not optimal. HiCRP’s highly selective binding behaviour suggests that it needs to make a greater number of direct contacts with the DNA to manifest a binding strength that EcCRP can accomplish with fewer interactions. In other words, more amino acids in HiCRP have to make specific base interactions and/or non-specific phosphate interactions with DNA for the protein to be able to hold on.

I have already identified a single mutation (lacZ-19A) that converts the lacZYA site into something that HiCRP can bind. However, HiCRP still binds to ICAP with >100-fold greater affinity than binding to lacZ-19A. Now it becomes a simple question of which bases in lacZ-19A prevent perfect binding by HiCRP? Six out of twenty-two bases differ between ICAP and lacZ-19A. Three of these differences are likely irrelevant or make minor contributions to binding, so I will ignore them at first. Of the remaining three, it is possible that no change alone will have a dramatic difference but that a particular combination of two key bases will have a dramatic effect. Thus, multiple possible permutations of mutated lacZ-19A sites arise, and it will be a lot of work to test all of them (so I won’t).

So, the simplest experiment is to make 3 single point mutations, but do I change ICAP to resemble lacZ-19A and measure the deleterious effects on binding, or do I change lacZ-19A to resemble ICAP and measure the positive effects? Because there is such a dramatic difference in HiCRP affinity for these two sites, I have good power to detect beneficial or deleterious changes. I am inclined to work with ICAP because I can measure the effect of each mutation while all other bases remain “perfect”. With lacZ-19A, I don’t know if the mutations I introduce would have greater or lesser effects on affinity if studied in the context of other naturally occurring CRP sites. The key question remains “which bases in a CRP site are most critical for HiCRP binding?” and not “why is the lacZYA site such a crap site for HiCRP”. Therefore, I will work with ICAP, and hopefully this will provide insight into the lacZYA issue.

CRP and Sxy plans

2007-10-01T20:21:00.000-07:00

Having completed all but a few final touches on the revised Sxy manuscript, I can now return my focus to the next most pressing piece of science/manuscript: an investigation into the molecular mechanisms by which CRP binds and activates CRP-S promoters. The initial (already submitted to and rejected by a journal) version describes experiments that compare how E. coli and H. influenzae CRP bind to DNA, followed by experiments examining how converting a CRP-S site to a CRP-N-like site influences CRP binding and transcription initiation, then some experiments testing Sxy dependence at CRP-S promoters, and finally experiments designed to test the influence of putative UP elements in CRP-S promoters.

This manuscript began life as a chapter in my thesis designed to pull together several good, but not very cohesive, experiments. New experiments conducted over the past few months have allowed this manuscript to evolve into a more thorough analysis of how CRP binds and activates CRP-S promoters. The constantly evolving nature has paved the way for some very neat experiments, however this means that the manuscript won’t be done for another couple of months yet. On the plus side, it will be a much more cohesive manuscript and the science will be more complete and exact (no more hodgepodge of results).

I now feel that the manuscript should be only about CRP and that experiments testing Sxy’s role in DNA binding and transcription activation should be saved for a Sxy-specific manuscript that combines my work with Sunita’s. I think Sunita’s excellent molecular biology skills are bringing us rapidly towards the ability to directly test how Sxy functions on a molecular level. Furthermore, I have recently acquired some V. cholerae DNA which I hope Sunita can use in her Sxy and CRP cross-species complementation experiments. Because very few regions of the Sxy protein are conserved between H. influenzae, E. coli and V. cholerae, if all three proteins work in all three species, this result will very quickly narrow down which regions of the protein are required for whatever function we discover Sxy to have. She and I will meet tomorrow to discuss our experimental and manuscript plans.

Measuring how tightly CRP binds to a perfect CRP-binding site

2007-09-18T15:00:00.000-07:00

It is important to know how tightly a transcription factor (or other protein regulator) binds to a specific target DNA site because this will dictate how long the transcription factor occupies that site. In the case of target sites in gene promoters, excessive affinity for DNA results in a loss of responsiveness to the signals transduced by regulatory proteins; for example, if CRP has an inappropriately high affinity for a gene promoter, it will continue to stimulate transcription potentially long after the cell has satisfied its energy needs. Conversely, if a protein has low affinity for a DNA site, it will move on and spend more time elsewhere on the chromosome. Therefore, all gene promoters are fine-tuned by natural selection to have a useful balance between protein binding and dissociation (except perhaps in intracellular symbionts, which can lose gene regulatory functions because they live in static environments).

Protein affinity for a DNA site is usually expressed as a dissociation constant, Kd (the lower case “d” should be a subscript capital “D”). A “dissociation” constant is used because it is technically difficult to measure how quickly a protein binds to DNA, whereas it is much more straightforward to measure how long it takes for a protein to let go after it has bound to a target site. Bandshift analysis (which we use in the lab) is the most popular technique for calculating Kd. The Kd value is expressed as the molar concentration of protein needed to bind, and thus shift, half of the radioactive “bait” DNA in a binding reaction. For example, 1 nM of CRP can shift half of the DNA when the mglBAC promoter is used as bait because this promoter contains a high-affinity CRP site. Therefore, for the mglBAC promoter, CRP's Kd = 1 nM. On the other hand, it takes 100 nM of CRP to shift bait DNA that contains a less favourable binding site.

In a bandshift assay, a purified protein (in my case CRP) is mixed with a low concentration of bait DNA containing a target site to which the protein is expected to bind, in addition to a very high concentration of "bulk" DNA for which the protein is expected to have very low affinity. Under these reaction conditions, the protein will spend much of its time bumping into DNA sites with which it can’t form lasting associations. Even though the protein will rarely bump into a bait DNA molecule, if it has a high affinity for a site in the bait DNA, it will hold on and not let go. If the bait DNA contains a low affinity site, more protein molecules are needed in the reaction so that as soon as a protein molecule lets go of the binding site, another protein molecule is likely to quickly bump into that same site and hold on, albeit for a short time.

I recently tested the binding affinity of both E. coli CRP and H. influenzae CRP for a perfect CRP binding site called “ICAP”. Both proteins demonstrated an identical affinity for the site. Because this was an unexpected result, further thinking revealed that the reaction conditions I used were not suited to detect super-high affinity. I realized that to detect super-high affinity, I would need to decrease the amount of bait DNA in my reactions. At the concentration of bait DNA used, protein with only a high affinity (as opposed to a super-high affinity) for the site could also readily shift half of the bait DNA. In other words, there was so much bait DNA in the reaction that I had to load more protein than should be necessary to shift even half of the DNA. This concentration of bait DNA has been convenient for testing low to high affinity DNA sites, but is not sensitive to super-high affinity. Thus, I will need to redo the experiments with vanishingly small (pM) amounts of protein and bait DNA.

Happy to have been wrong about HiCRP

2007-08-26T19:02:00.000-07:00

I conducted some very satisfying bandshifts this week, although I haven't found time to crunch and analyze the data. Thus, I will only be able to briefly describe my first impressions of the results. The first satisfaction came from confirming that my protein preps were very active in DNA binding; because preparing the proteins takes several days, I always live in fear through much of the preparation process that the final protein preps will be bunk.
The second satisfaction came from discovering that, contrary to earlier conclusions, H. influenzae CRP (HiCRP) appears to have a very high affinity for DNA, comperable to that of E. coli CRP (EcCRP). This revelation comes from testing both proteins' affinities for the theoretically optimal CRP-binding site, called "ICAP". In earlier experiments conducted in 2002 and 2005, I tested CRP affinity for a natural CRP site from the Hflu mglBAC promoter; this site was my positive control for DNA binding because it is very similar to the optimal site (ICAP). EcCRP bound the mglB site with very high affinity, but HiCRP did not. From this, I concluded that HiCRP just was not as strong a DNA binding protein. I hypothesized that HiCRP may be a weak DNA binding protein because it does not form very stable dimers (only homodimeric CRP molecules bind DNA). I think that my ICAP results may offer some support for this theory. Although HiCRP and EcCRP demonstrated a similar affinity for ICAP when they were in the 10-500 nM concentration range, my impression was that HiCRP binding was not detectable below 10 nM, whereas EcCRP binding could be detected down to 0.5 nM. In my thesis I lamented that I did not know of a good assay to detect dimerization of CRP molecules at the low concentrations where dimerization would be an important contributer to CRP's ability to bind DNA. However, bandshift assays are very sensitive to inter-molecular interactions at very low protein and DNA concentrations, so perhaps I will be able to glean some more informative data from my bandshifts than I had initially intended. This will require that I get a better handle on the calculations used and underlying assumptions made when measuring protein-DNA binding kinetics.

More bandshifts....

2007-08-20T19:00:00.000-07:00

The protein is pure and concentrated, the DNA is hot, and the scientific questions always abound, so tomorrow I start running bandshift gels (many, and perhaps more than humanly possible at one time). Fingers crossed for beautiful bands. Tomorrow, a blog about results (finally)!

Using bandshifts to study CRP-DNA interactions

2007-08-12T21:01:00.000-07:00

Along with cloning new pilABCD promoter constructs for testing the role of putative UP elements in CRP-S promoters (see July 30 post), I am purifying E. coli and H. influenzae CRP proteins to test their in vitro affinities for various gene promoters. I have been conducting similar bandshift experiments off and on throughout my graduate studies and have found some very curious and unexpected results (these unexpected results are undoubtedly part of the reason this work has been sporadic; it's sometimes very hard to know what to conclude and what to do next). In short, I found that E. coli CRP behaves, well, like E. coli CRP, which is all well and good. However, H. influenzae CRP (a highly similar ortholog with the same cellular role) exhibits dramatically different binding behaviour. For example, E. coli CRP has very high affinity and specificity for the lacZYA CRP-binding site, whereas H. influenzae CRP does not treat this site any differently from random DNA sequence. As a first step to figure out why, I have mutated the lacZYA CRP site to better match the CRP-binding site consensus sequence (only a single base pair change makes the lacZYA site nearly "perfect"). If this mutation is sufficient to change this site to a specific H. influenzae CRP-binding site, then we will know that the Hflu protein is highly selective for a specific base pair at position 19 in the 22bp site.

Peter, Peter, DNA eater

2007-08-07T08:47:00.000-07:00

During her research into the distribution of the competence phenotype in H. influenzae strains, Heather has discovered a strain, PittAA (which I call Peter), that demonstrates transformation frequencies 1000 fold greater than the reference strain (KW20) when cultured in rich medium. Specific mutations in the sxy and murE genes (and possibly also in crp) can cause similarly elevated transformation frequency in KW20, a phenotype we call hypercompetence. If we can track down the genetic cause(s) of PittAA’s hypercompetence, it may help us better understand competence gene regulation in our preferred organism, KW20. Can we track down a region of the PittAA genome that confers hypercompetence when transformed into KW20?

1. Is PittAA sxy responsible for hypercompetence?
Heather has described the amount of difference between the PittAA and KW20 sxy genes: http://heathermaughan.blogspot.com/. Unfortunately, we don’t know anything yet about how the Sxy protein works, so we don’t know if certain amino acid differences between PittAA and KW20 Sxy are functionally significant.

In KW20, mutations that destabilize the secondary structure of sxy mRNA cause hypercompetence. Examination of the homologous sequence in PittAA revealed three base differences compared to KW20: one in Loop C, one in Stem 3, and one in Stem 1A (see figure of KW20 structure). A base difference in Loop C is not expected to have an effect on 2º structure, and the differences in the stems conserve the base pairing potential seen in KW20 (C:G→U:G and A:U→G:U; differences between KW20 and PittAA are in bold). Therefore, non of these 3 sequence differences are expected to destabilize the mRNA 2º structure and thus do not readily explain the hypercompetence phenotype. Though unlikely, these base differences in PittAA may favour the formation of a different 2º structure than what we have characterized in KW20. I will see if the RNA-folding program Mfold predicts an alternate, but thermodynamically favourable, folding structure for PittAA sxy mRNA. One caveat is that we don’t know for sure whether transcription of the PittAA sxy gene initiates at the same base as in KW20; I will check for the position of the CRP site and RNAP binding sites in the PittAA sxy gene promoter and this should help us predict where transcription initiates in PittAA.

2. Is PittAA crp responsible for hypercompetence?
One possibility is that the PittAA CRP protein is constitutively active (ie. cAMP independent); in E. coli, multiple mutations have been identified in crp that make CRP constitutively active. To test whether PittAA CRP responds to cAMP, cAMP can be added to exponentially growing PittAA cells. Addition of cAMP is expected to increase transformation frequency, and if it does, we know that CRP activity is still limiting to competence gene induction during exponential growth.

The most exciting possibility is that PittAA has gain-of-function mutations in Sxy or CRP that allow one protein to act at CRP-S promoters in the absence of the other. If we could identify such a mutation(s), it would tell us a lot about how CRP and/or Sxy activate transcription at CRP-S promoters. To test whether PittAA has GOF mutations in CRP or Sxy, we can transform PittAA with KW20 crp- and sxy- constructs, but would have to use PCR amplified knockout genes in order to avoid transforming other parts of the PittAA genome. We predict that clean knockout mutations in PittAA crp or sxy will abolish transformation, as they do in KW20. If not, than a GOF mutation is the best explanation.

It is tempting to hope that only one genetic difference can explain the “non-KW20” phenotype in each H. influenzae strain that is non-competent, non-transformable, or hypercompetent. However, it seems likely that non-competent strains will have accumulated mutations in unused competence genes. On the other hand, a hypercompetence phenotype and a non-transformable phenotype can each be generated in KW20 with single mutations. Thus, it should be tested whether KW20 genes can "restore" the KW20 phenotype in the non-transformable strains.

Excited about UP elements

2007-07-30T09:34:00.000-07:00

Now that our "Regulation of Sxy" manuscript is submitted, I can shift gear and get back to working on the A/T rich sequences in CRP-S promoters that I hypothesize work as UP elements. First some background about UP elements: bacterial RNA polymerases make two specific contacts with promoter DNA, one is the well-characterized recognition of the -10 and -35 sequences by the sigma subunit, and the second is the less-familiar recognition of upstream A/T rich sequences (UP elements) by RNAP's two alpha subunits.

All of H. influenzae's 13 CRP-S promoters have A/T rich sequences resembling UP elements, and I have shown that these sequences are required for transcription of the pilABCD operon. My working hypothesis is that CRP and Sxy act at CRP-S sites to bend DNA and bring the upstream UP elements close to RNAP so that the alpha subunits can make contact with the UP elements. To test this I am going to remove the CRP-S site and some flanking sequence from the promoter, thus bringing the putative UP elements closer to the -10,-35 RNAP binding site. I predict that the pil promoter will then be active in the absence of either CRP or Sxy. In other words, because I have cut up the DNA and positioned the UP elements physically adjacent to the -10,-35 site, CRP and Sxy are no no longer needed to bring the UP elements close to RNAP by bending the intervening DNA.

Manuscript writing/polishing

2007-07-18T15:44:00.000-07:00

The latest round of experiments using T7 RNA polymerase in an E. coli S30 in vitro transcription/translation system has yielded very satisfying results. The results are so consistent with our model of sxy regulation that I couldn't have faked better data. Now I am integrating these latest results into our highly polished Sxy manuscript. This integration is fairly easy, but doesn't feel very satisfying (so my mind keeps wandering to my next project). Our manuscript has such an abundance of good data that my new results don't tell us anything new, instead they simply reinforce our old model of sxy regulation. Let's hope that the one reviewer that has been holding out for these results will be satisfied.

Success with T7

2007-07-12T17:51:00.000-07:00

During the past few days I have conducted an experiment to test whether cooling T7 RNA polymerase to 25º allows sxy gene transcripts to fold in vitro as they do in vivo. Although I have not directly tested whether sxy transcripts are folding into the structures we have previously determined experimentally, conducting in vitro transcription/translation at 25º yields results much more in line with what we have detected in vivo. The new results are that sxy-1 is translated at high levels, whereas wildtype and sxy-7 transcripts are not. I am currently repeating the experiment and am also allowing the reactions to proceed for up to 4 hrs to see whether a difference emerges between the two poorly-translated transcripts (WT and sxy-7).

Trying to make T7 RNAP behave like bacterial RNAP

2007-07-08T21:58:00.000-07:00

I have been using coupled in vitro transcription/translation to test whether the secondary structure at the 5' end of sxy transcript blocks translation. Our strong genetic and in vivo results suggest that the sxy-1 mutation weakens 2º structure and so facilitates ribosome binding; the sxy-7 mutation does the opposite.

My in vitro results show a very little bit more translation in sxy-1 relative to wildtype, and about half as much translation with sxy-7. This trend is nowhere near as dramatic as the in vivo results, so Rosie and I have been troubleshooting the in vitro assay in an attempt to make the test tube more like in vivo conditions. A central complication is that the the in vitro assay uses the phage T7 RNA polymerase to achieve high levels of transcription. T7 RNAP is a very fast and processive (ie. it never drops the ball) enzyme. T7 RNAP transcribes about 10 times faster than E. coli RNAP, and this high-speed can result in miss-folding of RNA (Lewicki et al. (1993) showed this by using T7 RNAP to transcribe the 23S rRNA gene: the T7 product was non-functional, but melting and renaturation restored 23S functionality). Yesterday, I repeated the in vitro experiments at 25ºC because Lwicki found that this low temperature slowed T7 RNAP to a speed similar to E. coli RNAP.

We were also concerned that using circular templates for transciption might result in poly-cistronic transcripts; in other words, T7 RNAP may continue to transcribe around the plasmid template and never stop, resulting in a potentially miss-folded string of sxy genes. To test whether this is a problem, I linearized some of my templates before transcription/translation. I will have the results by Thursday, and our modified proceedures may yield results more like our in vivo data.

Back in the lab

2007-07-04T09:34:00.000-07:00

Now that I'm back in the lab, I have two primary goals: 1) Finish the experiements required for our Sxy manuscript, 2) Conduct a few more experiments to beef up our CRP-DNA binding manuscript. For #1, I will again assay the translatability of various sxy gene mutants. This is a replication of an earlier experiment so it should go smoothly. Preliminary results support our model in which strong sxy 2º structure reduces translation; if I confirm these results, the manuscript is done. I may include an experiment to test whether a short DNA oligo can compete with formation of sxy 2º strucutre and thus facilitate translation of sxy mRNA. I attempted this experiment before, but found that the short mRNAs I was using encode a half size Sxy protein that is not detected by our anti-Sxy antibodies. I can now repeat this experiment using my new clones that generate full-length sxy mRNA.
For goal #2, Rosie and I may decide to resubmit the CRP-DNA binding manuscript as is, or I can conduct a few more experiments to improve it. Today I will write a draft of the proposed experiments so that we can weigh our options.

"It's been a long time (since I rock and rolled)" - Robert Plant

2007-04-02T15:03:00.000-07:00

A former member of our lab found that adding adenosine monophosphate (AMP) or guanosine monophosphate (GMP) to MIV or sBHI culture media greatly reduces transformation frequency in H. influenzae (Macfadyen et al., 2001, Mol. Micro.). How do nucleotides down-regulate competence in H. influenzae? An earlier candidate for mediating this response was the PurR protein that represses genes for purine biosynthesis when intracelleluar pools of guanine or hypoxanthine (a precursor of AMP and GMP) are high. Thus, in this early model, PurR blocked the expression of purine biosynthesis genes AND competence genes; however, knocking out purR did not result in elevated competence. Bioinformatic analysis still implicates PurR in the regulation of competence because the rec2 gene has a near perfect PurR binding site in its promoter. This leads to a model in which PurR plays only a limited role in regulating competence, which may be tricky to detect in the laboratory conditions used to study competence.

In collaboration with a former member of the lab, we found that transcription of the sxy gene is reduced in MIV+AMP, resulting in a dramatic decrease in Sxy protein levels. However, cells also suffer general malaise in the presence of these high concentrations of nucleotides, thus we need to repeat these experiments with controls for deleterious effects on the expression of non-CRP-S regulated genes. In other words, we can’t say for sure that nucleotides repress competence due to a specific effect on sxy expression. I recently measured the levels of Sxy protein in hypercompetent strains and found that even though cells have very high levels of Sxy protein, they experience the same repressing effect as wildtype cells. This raises three possibilities: (1) Nucleotide repression arises simply because cells are sickly in nucleotide-supplemented media, (2) An as yet unrecognized factor specifically represses competence in the presence of nucleotides, or (3) Sxy has a nucleotide allosteric effector. In (3), nucleotide binding to Sxy is expected to prevent the protein from acting at CRP-S promoters, in contrast to how purine bases cause PurR to bind DNA.

Possibilities number (1) and (2) can begin to be addressed by measuring the effect of nucleotide supplementation on the expression of genes that do not require Sxy for expression (possibility number (3) requires the same controls to know that we are testing effects on Sxy). For (3), we can now test the effects of nucleotide supplementation on competence gene expression in an E. coli strain that overexpresses sxy; this is much like the H. influenzae hypercompetence mutants, so we might see that competence gene expression is repressed even when sxy expression is high. If so, it may be worth testing if Sxy binds to nucleotide-coated resins.

What got me thinking about whether Sxy has an effector molecule was my latest BLAST search for recognizable protein domains. The result has been the same for the last five years: the top hit that isn’t a Sxy ortholog is the weak alignment of Sxy’s C-terminal half to a cytidine deaminase from N. meningitidis (e value 0.006). Cytidine deaminases hydrolyze cytidine into uridine. Unfortunately, this alignment does not appear to be informative because it is questionable whether the N. meningitidis protein is in fact a cytidine deaminase (as annotated). TIGR calls this gene “cytidine deaminase”, but TIGR’s “identification alignment” is with a hypothetical ORF of unknown function in B. subtilis, and warns that it is only an automatic annotation. The N. mening ORF does align with a portion of the zinc binding domain of the cytidine deaminase family of proteins, but this is not the same region that aligns with Sxy. Unfortunately, there is nothing published about the N. meningitidis ORF.

Competing stems?

2007-01-16T14:35:00.000-08:00

In preparation for a manuscript describing how the sxy gene is regulated in H. influenzae, we want to illustrate predictions of how sxy mRNA folds into stems and loops. A very useful program called Mfold can generate loads of pictures illustrating predicted secondary structure (ie. 2-dimensional representation of the stems and loops predicted to form in an RNA molecule). In addition to these predictions, we have experimental analysis of sxy mRNA secondary structure. Ultimately we want to compare and contrast Mfold's thermodynamic predictions with the experimental data of sxy mRNA folding.

However, we have a problem. Because RNA molecules are dynamic and can often fold into many alternate secondary structures that are all thermodynamically equally (or almost equally) stable, Mfold provides multiple predictions for a single RNA sequence. Comparing the data with predicted structures is all well and fine at positions where we have data, unfortunately the structural data is patchy, thus we lack experimental insight into some regions of the sxy molecule. Consequently we are left with the problem of deciding which Mfold prediction is “best” for these experimental blind spots.

Experiments confirm the prediction that the 5’ end of sxy mRNA folds into what we call Stem 1A and Stem 1B. What we don’t know is whether Stem 1 also has a third region (Stem 1Z), or whether an adjacent Stem 4 forms from some of the same sequence. Figure 1 shows two alternate Mfold predictions, Structure 1 with Stem 1Z and Structure 2 with Stem 4 (the two structures differ in sequence length, but that difference is not important for this discussion). The bases that contribute to Stems 1Z or 4 are highlighted green to show that the two stems are mutually exclusive.

In the absence of experimental data for the green sequences that form stems 1Z or 4, I have tried to estimate the relative probability of sxy forming either stem by comparing multiple Mfold predicitons. I asked Mfold to predict the best structures for the 340 nucleotide sxy RNA molecule used in the experimental structural analysis. It returned 25 predictions with a Gibbs free energy (delta G) between -75.5 and -69.2. Of these predictions, both stems showed up in multiple structures across the full spectrum of this free energy range, indicating that neither stem contributes to a more thermodynamically favourable strucutre. However, Stem 4 (including variants that have longer and shorter Stem 4s with different loops at the end) formed in 8/25 structure while Stem 1Z formed in only 4/25 structures. The real surprise was that 14/25 structures looked nothing like our favourite model of sxy folding presented here (Figure 1). In these alternate structures, the green sequences are as diametrically opposed as possible on the large RNA molecule (an example is in Figure 2). These alternate structures do not immediately appear to agree with our genetic evidence of sxy mRNA folding, so we can ignore them for now. However, counting the frequency of stem formation in Mfold predictions has got us no closer to deciding on whether to draw sxy mRNA folding with Stem 1Z or Stem 4.

A note on today

2006-12-20T09:51:00.000-08:00

Hey Blog. Today I am composing responses to questions that have arisen in Rosie's current grant proposal. I will also describe some experiments that may be of use in the proposal.

2006-12-03T18:36:00.000-08:00

I am currently conducting what I think to be a cool experiment to turn the E. coli lac promoter into a Sxy-dependent (or possibly Sxy-enhanced) promoter. Earlier this year I received a very useful clone, pASKAsxy, from a group in Japan; it carries the E. coli sxy gene under strong LacI repression. Using this clone I discovered that Sxy induces E. coli competence genes, but not genes with normal CRP-N sites. Now I want to test whether it is the TGCGA motif of CRP-S sites that single out CRP-S promoters for activation by Sxy. In other words, does having the TGCGA motif make a promoter Sxy-dependent?

I have mutated the CRP site in the classic lac promoter (Plac) to change it from the CRP-N motif (TGTGA) to the CRP-S motif (TGCGA), which I call “Plac-S”. My hypothesis is that Sxy will strongly induce Plac-S, but not regular Plac (possible results are discussed below).

But first, a technical problem. Plac and Plac-S are each carried on the pSU20 vector that confers resistance to chloramphenicol. pSU20 uses the p15 origin of replication and is compatible with the pASKAsxy ColE1 origin, however pASKAsxy also confers chloramphenicol resistance. To remedy this antibiotic incompatibility I have excised Plac and Plac-S from pSU20 while at the same time removing Plac from pSU40, a close relative of pSU20 that confers kanamycin resistance. Today I cloned the pSU20-derived Plac and Plac-S into pSU40. Next I will clone both pASKAsxy and pSU40-Plac concurrently into DH5a.

I can’t do a simple +/- sxy experiment as I have done in the past. The problem is that both sxy and Plac are LacI repressed, thus in the sxy- state (ie. no IPTG), Plac can’t be activated anyway. Thus, IPTG will always have to be present to detect differences in Plac induction levels. To control for sxy activity I will also clone pASKAcrp and pASKAyrfD with pSU40-Plac; the former clone is expected to stimulate Plac but not Plac-S, while the latter (which encodes the E. coli comA homologue) is not expected to activate either Plac or Plac-S (see Table). I will quantify Plac activity using real-time PCR directed at lacZ-alpha subunit mRNA. DH5a lacks lacZ-alpha, so the signal will only come from plasmids.

Discussion

2006-11-22T09:30:00.000-08:00

Today I tackle the sxy manuscript discussion. We still have a little more work to do on the RNase data/figure/results section, but otherwise it is only the discussion that needs to be massaged from notes into a coherent flow. The next draft of the manuscript should be done by the end of today or tomorrow - watch out lab mates, it's editing time.

In non-science news, I spent nearly an hour and half yesterday in my continued search for an AC/DC adaptor for one of our weigh scales. The dead adaptor has a highly unusual combination of voltage, amperage, and polarity but I did eventually find a replacement - unfortunately, it comes from China and we must purchase at least a 1000 units. Amazingly, the units are only $1.25 each. This a clear example of the rape of China where a sizeable piece of electronics, wiring, plastic, and resulting pollution is valued so low yet still includes a profit for the manufacturer. On the plus side, we can have it labelled with any company name we want, so it could be a custom "Redfield lab" power supply.

A sexy new machine

2006-11-09T16:50:00.000-08:00

Yesterday I ran an experiment to test whether my failed Friday the 13th real-time PCR was due to bad master mix or a bad machine. I ran a very simple set of reactions using a dilution series of chromosomal DNA as template. Reactions were run with old or new master mix on the new real-time PCR machine in the lab next door. All primers generated nice amplification plots regardless of which master mix was used, thus my Friday the 13th problems were caused by our old machine. Today I have repeated my experiment from last month, but data analysis is going slowly as I learn how to use the new analysis software. Even with the frustrations that often arise when learning one's way through fancy new software, I am generally impressed with the newer machine and software. In the 5 years since our previous analysis software was developed, data visualization and interactiveness has come a long way. Now that I've had my coffee, it's time for another session of real-time analysis.

Opposite to expectations

2006-11-06T16:09:00.000-08:00

In past blogs I have alluded to some analyses where I compared sxy mRNA and protein abundance between strains carrying mutations in sxy. I found that hypercompetent sxy mutants produce on average about twice as much transcript but 10-50 times as much protein as wild type cells. In other words, a sxy transcript in a hypercompetent mutant is translated much more efficiently than a transcript in a normal cell. Thus, sxy appears to be post-transcriptionally regulated

I thought these results may be telling us about how sxy expression is regulated in normal cells. I predicted that transferring cells from rich to starvation medium would result in a slight increase of sxy promoter activity and a large increase in translation of sxy. I have crunched the data and, very surprisingly, found the exact opposite result. Upon transfer to MIV, sxy transcript levels immediately skyrocket by 25 fold, then quickly return to pre-induction levels. Protein levels show a much more gradual increase to about 10 fold pre-induction levels, followed by a slow decline.

This result is consistent with what we know about the relative stability of the mRNA and protein – the former is generally very short lived and the latter is long lived. However, it means that cells do not use post-transcriptional regulation to induce sxy upon transfer to inducing conditions – or, if they do, the effect is masked by a very strong inducing signal acting at the sxy promoter. The result may also indicate that in MIV sxy transcript half-life is insufficient to allow significant translation, hence the only moderate increase in protein level. In other words, it is the opposite of what I described above. In a hypercompetent mutant in rich medium, one transcript can make a lot of protein, whereas in wild type cells in MIV, some transcripts may be degraded before even making one complete protein.

Fluorescence is the problem

2006-11-03T16:00:00.000-08:00

It turns out that the problem with my real time PCR run on October 13 is something to do with fluorescence. In the last post I described how I didn't see any amplification, but the fluorescence levels appeared abnormally high from start to finish. Today I ran the PCR products out on a gel and found that the PCR worked very well, as expected with my well-used primer sets. The products were clean and lacked any primer dimers. So, if the PCR worked, it is either the fluorophores in the reaction mix that are bad or the machine is broken and doesn't detect them properly.

There are two fluorophores in the reaction mix we use: SYBR and ROX (newer machines don't use ROX). SYBR is the dye that binds to DNA and is used to track amplicon abundance between PCR cycles. Rox, on the other hand, is a passive dye that doesn't interact with anything the reaction. ROX and SYBR have non-overlapping excitation and emission spectra. Thus, the machine can idenpendently read the SYBR and ROX signals: the SYBR signal is expected to vary dramtically between wells and change throughout the PCR run while the stable ROX signal is used to correct for variance that arises because of differences in transparency between wells.

My next step is to compare our old Master Mix with the new suspect batch. I may run this experiment on the new machine next door to make sure it is not our old machine that is the problem. However, this requires learning to use a new machine and analysis software and burrowing PCR tubes from the machine's owner.

Back in the lab

2006-10-31T13:23:00.000-08:00

I am currently planning my experiments for the next couple of months so that I know exactly what I want to be doing each day. My first goal is to resolve a problem that arose just before my departure to Inuvik. I was repeating an experiment to quantify the relative levels of sxy mRNA in strains that have regulatory mutations in the sxy gene.

There are several steps required to do this. First, cells are cultured and all RNA is extracted. The RNA samples are treated with DNase, then random hexamer-primed reverse transcription is used to convert the RNA into cDNA. The cDNA is then used as template in a SYBR green real time PCR assay. In this assay, more starting template results in the synthesis of more amplicon early in the PCR cycles. Amplicons are bound by the SYBR green fluorophore and fluorescence increases; fluorescence is measured each thermocycle. There are many steps where problems can arise, but appropriate checks and controls are used throughout. First, a gel is run to test the quality of the extracted RNA. Later in the final PCR reactions, non-DNase treated RNA is included as a template to ensure 1) that there is more template in reverse transcription reactions, confirming that the RT step worked, and 2) that chromosomal DNA is not a significant contaminant. Also, chromosomal DNA is used to confirm that the PCR Master mix can amplify target genes.

In the past we have had problems with chromosomal DNA contamination during the RNA extraction/purification, but that has long since been remedied. On Friday Oct. 13, I encountered a whole new problem: no amplification was detected in any of the real time PCR reactions. One odd result is that the fluorescence levels were very high throughout the run, suggesting that there may have been amplification as usual, but that the Master Mix fluorophores or the detector are not working properly. There are other possible suspects: 1. The least probable problem is that the primers have gone bad (I know they were added to the Master Mix), but they worked beautifully a couple of months ago. 2. I used a new batch of SYBR Master Mix, and it may have bad Taq enzyme or bad fluorohpores. 3. The machine is not thermocycling or its detector is not working properly.

My first troubleshooting step is to run some of the PCR reactions on a gel to see if amplification did occur. If yes, fluorescence is the problem: either the detector or the MMix is not working. If no, Taq or the thermocycle is not working. Either way, the second step will be to run a couple of reactions comparing the new Master Mix to older leftover MMix. Also, I will include another primer set to test whether my primers are the problem. If I again see no amplification, I will try the reactions again on a different realtime PCR machine.

On the positive side, my cDNA samples are already prepared. Once I solve the PCR problem, it will be relatively quick to measure sxy mRNA levels as I had originally set out to do.

Yesterday's graph is wrong

2006-10-24T05:58:00.000-07:00

I awoke early this morning with a nagging feeling that I had made a bad graph yesterday. The second graph in "Musing on Sxy", which outlines my predictions about how adding cAMP to log-phase cells will affect transformation frequency, is missing data we already have. What would help is if my imaginary graph became more concrete with some actual numbers and data (again from Redfield 1991). First, a reminder that our simple model for the regulation of competence currently involves two activators (or two “on” switches), CRP and Sxy. CRP levels are fairly constant throughout different growth stages; by supplying 1mM of it’s allosteric effector cAMP, we are turning that switch full on.

This graph is now based on real data - the four data points are roughly the average transormation frequencies measured in Redfield 1991. Here we see moderate competence development in KW20 when CRP is fully activated, even though KW20 has very little Sxy during log-phase growth. Thus even very low levels of Sxy are sufficient to allow some competence, in contrats to what I said yesterday. Also, this helps clarify what I was already planning for today’s blog: I want to take a reciprocal approach to yesterday and look at what limits maximal competence when both the CRP and Sxy switches are turned on. (A subsequent blog will discuss the role of stochasticity and competition/interference between regulators - but that’s trickier and needs clear groundwork thinking. I’ll stick with the simple on/off switch analogy for now).

Maximal competence (ie. a transformation frequency just over 0.01 with good, fresh transforming DNA) develops when cells are incubated in MIV medium for 90 minutes. Redfield 1991 found that the sxy-1 hypercompetent mutant achieves the same high transformation frequency in MIV and during late log-phase growth. When I repeated this experiment to test all hypercompetent mutants, I observed a ~5 fold lower maximal transformation frequency in MIV , and transformation frequency in late log was 7-10 fold lower again (see graph). However, I used old transforming DNA and found that with fresh DNA, transformation frequencies in MIV were the same as Redfield 1991 (not shown on graph. Also, I did not test late log growth with fresh DNA). This raises the possibility that in Redfield 1991, using very potent, fresh transforming DNA obscured a difference in transformability between MIV and late log conditions, which only became apparent when using old (sub-saturating?) DNA. If so, why don’t hypercompetent sxy mutants achieve maximal transformation in late log-phase? One possibility is that the cells are maximally competent, but the transformation machinery within cells is more efficient in MIV conditions. However, I will explore the alternate hypothesis that hypercompetent sxy mutants are not maximally competent in late log growth.

Hypercompetent sxy mutants in late log-phase have equal amounts or more Sxy than does KW20 when it is maximally competent in MIV, therefore Sxy is not limiting. The cAMP data described yesterday and at the beginning of this post shows that cAMP is not limiting. Thus, I can elaborate the first graph, and include a possible role for a third regulator. We have good evidence that PurR, a protein that represses gene expression in the presence of the purine bases hypoxanthine or guanine, is a third regulator of competence. To date, we have been testing for PurR activity by supplementing growth media with purine nucleotides, but this has been problematic partly because cells don’t like having high levels of these nucleotides in their growth medium. If all of my speculation presented here has some validity, we can test for PurR activity in late log-phase by measuring transformation frequency in double hypercompetent sxy/purR- mutants. If the hypothesis is correct, then when transforming with sub-saturating DNA, hypercompetent purR- cells will have higher transformation frequencies than their hypercompetent parents.

Musing on Sxy

2006-10-23T11:55:00.000-07:00

In my last blog posting I said that both Sxy and cAMP-CRP are limiting to competence development during growth in rich medium. However, I would like to make a more precise statement about how these limitations may change as culture conditions change. Here I expand my previous graph showing the correlation between increasing Sxy levels and increased transformation in both KW20 (blue) and hypercompetent mutants (red) during log (OD 0.2; filled circles) and late log (OD 1.0; empty circles) growth in rich medium (see "A strong positive correlation"). The black line plots the correlation, and the dashed black line is a hypothetical leveling-off of transformation frequency when Sxy levels become saturating. The dashed green line is a prediction of how the plot would look if cAMP were added to the culture medium.
Redfield (1991) showed that adding cAMP to hypercompetent sxy-1 cells increases transformation frequency in early log but has no effect in late log growth. Therefore, cAMP is not limiting to competence development at ~OD 1.0. The green line is not predicted to have the same slope as the black line because cAMP is only limiting during log growth; presumably, as PTS sugars are depleted in late log growth, cells are making their own cAMP. Thus, the two lines converge and level-off when Sxy becomes saturating. We know that Sxy isn’t saturating during late log because some hypercompetence mutants have more Sxy than others, and this correlates directly with increased transformation frequency.

This predicts that titration of cAMP during log growth will have no effect in KW20, but will increase transformation frequency in hypercompetent mutants such as sxy-1.
This hypothetical graph shows that due to insufficient Sxy levels in KW20 during log growth, no competence develops even at high cAMP levels. On the other hand, there is sufficient Sxy in sxy-1 to activate competence genes, thus adding cAMP increases transformation frequency. Hypercompetent strains are transformable in log growth, so there must be some cAMP produced by cells in rich medium. In the graph, the cyaA- mutants cannot make cAMP thus even the sxy-1 cyaA- double mutant cannot become competent until cAMP is added.

A strong positive correlation

2006-10-19T21:26:00.000-07:00

My time in Inuvik so far has consisted primarily of making figures. In order to stop procastinating, here's a blog post to show a satisfying result. (A "Where is Inuvik?" post will follow soon). Plotting Sxy abundance in cells growing in sBHI against transformation shows a positive correlation: as Sxy levels increase, cells become more competent.
This tells us that Sxy is limiting to competence development during gowth in rich medium, as is cAMP-CRP. In the figure, blue=KW20 and red=hypercompetent mutants, while filled circles are OD 0.2 and empty circles are OD 1.0. This plot includes our latest protein data - with this new data, the corellation between Sxy levels and transformation frequency is very strong (and satisfying).

"A quick one while he's away"

2006-10-09T19:37:00.000-07:00

The title is an homage to The Who, who it turns out have just released a new single after all these years, but refers more to the state of my brain while writing this post. And this has just reminded me of an article I skimmed over in (an old) Nature today describing how new neurons in adult brains only become established if they are stimulated. Thus, our everyday activities and thoughts get hardwired to a certain extent by selecting for neuronal pathways that contribute to or fit with our brain processes that day - budding new neurons that aren't stimulated die off. So Cliff Claven was right that there is selection for neurons in our brain, though not as he imagined it (ie. beer kills slower and weaker neurons, thus making the remaining herd of neurons faster and the brain smarter... I think he explains it with an analogy of lions killing sick and old wildebeest).

This weekend I had hoped to repeat some old experiments conducted by a former grad student in the lab, however there are no freezer stocks of the strains he made, and worse yet, used. In other words, he used up the last of some older strains in the lab. I bow my head in sorrow at the extinction of some unique and special bacterial species. Oh well, I'll just have to make them again. Also this weekend, I almost wrapped up my NSERC application - that will easily be done tomorrow. On the dock this week: review a manuscript, measure some sxy mRNA, start to put together a manuscript on CRP binding to CRP-S sites, make some strains and repeat the old experiments to test whether PurR regulates rec2 expression, and probably all sorts of other odds and ends (or, "Odds and Sods" as The Who would say)...

"On it's way..."

2006-09-28T15:42:00.000-07:00

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.

Good news Band news

2006-09-26T17:16:00.000-07:00

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.

Spankleen?!

2006-09-19T21:25:00.000-07:00

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

Nalgene ≠ plastic

2006-09-18T10:44:00.000-07:00

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.

Gels/manuscript/PurR day

2006-09-12T09:08:00.000-07:00

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.

A day of revisions

2006-09-09T13:00:00.000-07:00

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.

No background

2006-09-07T08:13:00.000-07:00

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.

Yesterday's blog today

2006-09-06T09:25:00.000-07:00

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.

A rushed post after another drought

2006-09-01T13:57:00.000-07:00

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.

Been a while...

2006-08-28T19:24:00.000-07:00

And, sadly, after an exhausting day I'm not going to post much to make up for it. Tomorrow is dedicated to composing pieces of our sxy manuscript, including: crunching today's data, getting figures together, writing my and Laura's Material and Methods sections, finishing the strain list, and (time premitting, which I doubt) thinking about elements of the discussion.

Now, a little bit of thinking on MurE hypercompetence mutants: Inspired yesterday by handing Rosie a figure showing that addition of AMP or GMP to MIV cultures results in reduced Sxy production. A neat feature of the data is that AMP has a more dramatic effect than does GMP on Sxy levels, and this is exactly what we see in terms of repression of transformation frequency. It got me to thinking that I saw the same thing when treating murE749 cells with nucleotides, which got me wondering if murE749 induces competence through the standard Sxy + CRP mechanism, does it do so by specifically inducing sxy? Caixia describes testing one of Laura's sxy::lacZ fusions in the murE749 background, but the strain described in that paper is misslabeled in the paper's strain list (ie. our records show the strain number from the paper to correspond to a xyl mutant of some sort). I sifted through Caixia's notebook and could find most of the other murE experiments, but not this one... So I don't know what type of fusion Caixia tested: protein or operon? It's possible she tested an operon fusion, which we now know doesn't acurately reflect Sxy abundance in the cell. I could directly measure Sxy levels in the murE749 strain, and if we found that they were increased, we could be fairly confident that murE749 induces competence through Sxy. This would be interesting, but also just another piece of the complex and unresolved story of how sxy is regulated.

RNA vs protein

2006-08-23T18:49:00.000-07:00

The results are in: sxy transcript levels vary much less dramatically between mutants and between growth conditions than do Sxy protein levels. This suggests that RNA secondary structure has it's greatest effect post-transcriptionally by influencing translation.

Yesterday's real time pcr experiment gave nice results consistent both with expectations (ie. more RNA in hypercompetent mutants and less in "hypocompetent" mutants) and with microarray results (ie. RNA levels increase in WT cells as cell growth slows at the onset of stationary phase). Today I have been crunching yesterday's RNA results, reanalyzing old protein data, and learning to use the new fluorescence scanner in our department (which will be used this week for western blot detection). Tomorrow I start the latest round of western blots, and then some beautiful gel pictures for our manuscript.

RNA becometh DNA day

2006-08-21T08:46:00.000-07:00

Today I destroy some DNA and make some more (ie. DNase treat my RNA samples and then reverse transcribe them). I can then do the real time pcr tomorrow, and then western blots to measure Sxy levels during the rest of the week. I am very eager to get this sxy manuscript together, so I will bust my ass to get the results we need before the long weekend. At the same time, I'm going to work more on writting the manuscript - it's hard in the absence of the key experiments I'm doing now, but I will try write outlines to address the possible outcomes and begin to flesh out the discussion. I look forward to writing the discussion because it will require delving into some good ol' RNA regulation work from the microbiology community.

Also, if my real time pcr shows that hypercompetent mutants have higher levels of full-length sxy transcripts, I want to draft up an experiment to test whether WT cells generate just as much 5' UTR RNA, which would suggest that the the hypercompetence mutations allow for full transcript reads, while in WT the transcript is prematurely terminated by formation of the loop. The outcomes of this experiment could be complicated by many things (ie. rapid degradation of 5' ends when they are not followed by a full transcript), so I hope to mine the trp attenuation literature to see how that model was established. A full discussion of attenuation will also be necesarry for the discussion of the paper as we work to solve how sxy is regulated.

Cell lysis (or lack thereof)

2006-08-18T08:56:00.000-07:00

My poor RNA recovery has resurfaced, and it is exclusive to cells in MIV for 30minutes, with improved (but still lower) yields from cells in MIV for 90minutes. I went back through my old notes to find that previously when working with Hflu, I had speculated that MIV treated cells require more intense lysozyme treatment. For many months I have been working with E. coli and had continued on with the same protocol when switching back to Hflu, with the standard thought that "Hflu is a wimp - it falls apart if you look at it long enough". But maybe I'm wrong, maybe MIV treated Hflu is a tough nut to crack. I have been visually cheking my cell suspensions for clarity, which is usually a good indicator of lysis, so I really don't know what's up.

Today I will add more lysozyme and also split my pellets so as to load less cell material onto the RNA isolation columns. It's possible that a tough cell wall is not the problem, but that something else about the cell constituents of MIV treted Hflu is negatively impacting column performance. By diluting the cell prep and loading less material, I hope to dilute the inhibitor and ultimately improve RNA yield.

I had a good visit with the Sigma rep yesterday. We really like their DNA purification and isolation columns (half the price and twice the performance of Qiagen = 4x better), and he said they now have RNA isolation columns, so he's sending a sample for us to try. It turns out that Sigma makes the buffers that Qiagen sells in their kits, so it's not surprising that Sigma can make a column and undersell their competitors (who they also sell to)... Sigma really can't lose.

More RNA isolation

2006-08-17T08:53:00.000-07:00

Since submission of the CRP-S manuscript Monday evening, the past two days have been spent back in the lab. A fair amount of sorting, tidying, ordering, cleaning, and other boring stuff - but the tedious RNA preps should be done today. I had poor yield from two samples yesterday and there is no rhyme or reason to their poor quality. The cell pellets were ample in size and they were prepared concurrently with good yield samples. I'm tempted to blame Qiagen for poor column consistency. Their name usually strikes a sour cord with me as they sell very expensive stuff, yet have some of the most useless tech support. To Qiagen's credit, even with low yields, the RNA still looks great and the sample is not infiltrated by RNases. Hopefully, better yields will result today.

Aleeza successfully defended her thesis yesterday. She did well, and I took mental notes to try and remain as relaxed and composed as she when my own defence comes around. I was particularly frustrated by two things: 1) the audience gets their only chance to ask questions immediately following the intro presentation, while their minds are still swirling from the tonne of info just presented. Instead, the audience should get to ask questions after the committee (who have all just read the thesis) ask their paced and thought-out questions. I think that this would do much to stimulate the audience into asking more pointed questions. 2) (this is not to be missconstrued as sucking up to the boss) The questions were all the same and rather boring. They were exactly what you would expect based on the material presented. Defences benefit from Rosie-style questions, which probe different and accessory (but equally relevant) issues. At least the examining committee did sound genuinely interested in the thesis material, even if they all fixated on the same stuff.

Manuscript is done!

2006-08-14T08:45:00.000-07:00

Well, besides a few i's that need dotting. Unfortunately, it took the weekend, mostly to get the citations all sorted (77 in total now) and the citation manager Bookends to put them all together. This means that this afternoon I can get down to sxy - both some RNA work and filling in some pieces (like Methods) in the 2º structure manuscript. Maybe soon I'll have something more scientifically stimulating to add to this blog - I think my brain was bored this weekend doing menial things like replacing ( ) with { } at all citations in the CRP-S manuscript.

Of scientific interest, may I didrect your attention to a recent article in Science "Depletion, Degradation, and Recovery Potential of Estuaries and Coastal Seas" (23 June, p. 1806-1808), which tracks human impact on coastal ecosystems across the ages of human expansion (the cultural periods make for a neat X-axis). There is currently much press on estimating the future impacts of global warming, but what strikes me is how we rarely stop to think about how our everyday activities, like fishing and pissing in the river, have, over the ages, caused more global damage in terms of depletion of species richness and abundance than global warming is predicted to accomplish in the next few hundred years.

Manuscript be done

2006-08-11T08:48:00.000-07:00

Today I get the CRP-S manuscript done.
1. Finish figure legends, 2. Finish table of contents and strain list, 3. Get bookends working, 4. Make sure everything is up to NAR snuff, 5. Finish fig.8 (make a nicer tree - include sxy evolution) 6. Do a final read-through... Then, on to Sxy - and I can commit myself wholeheartedly to studying "the other" regulator.

On a sad note, today I have a final visit with a dear friend (and co-founder of our long-running Darwin reading group) before he moves off to the States to work for the Department of Home Land Security (or at least be funded by them) to model the spread of terrorist seeded diseases. He comes from a long line of politically active communists, which meant a childhood of moving great distances and living in exile. He's had trouble visiting the states before, but now they want him to come work the front line against a new evil breed of people. But what happened to communists? Aren't they (and their children) still evil? Did Patrick Swaze make Red Dawn for nothing? If I were a super bad guy, I don't think I'd like it if my fickle nemesis decided to drop me from the radar.

2006-08-10T09:26:00.000-07:00

Today is an RNA day (with a few dashes of manuscript work thrown in for flavour).

The Sxy protein is required for the induction of competence genes in H. influenzae. We are working to dissect the mechanisms by which RNA 2º structure regulates sxy expression, to ultimately identify the signals that stimulate competence. I have already found that mutations that destabilize sxy RNA 2º structure result in increased Sxy protein production. Is this because the mutations allow for increased sxy transcription or increased translatability (or a bit of both)? I have prepped cell pellets from sxy mutant strains and now I will extract the RNA (26 samples in all)... tedious, but the results from quantifying sxy RNA levels will be most rewarding.

In the beginning...

2006-08-09T10:14:00.000-07:00

Hello lab gang,

I was hoping to support UBC's blog host, but it's not particularly well maintained and it's much easier to use this Blogger site.

Before the science of the day, an intro to my site:

"Duckfeet" and "Duck in the muck" are both references to the observations and experiments described in Darwin's "On the origin...". Darwin, in his supremely subtle brilliance, draws parallels between his simple experiments with ducks in a pond and the biogeography of plants and small animals that range across and between continents. I love this passage because it is both brilliant and funny. I'm including it here, but as I'm working in Safari, I can't readily use formatting to highlight it.

"Although the beaks and feet of birds are generally quite clean, I can show that earth sometimes adheres to them: in one instance I removed twenty-two grains of dry argillaceous earth from one foot of a partridge, and in this earth there was a pebble quite as large as the seed of a vetch. Thus seeds might occasionally be transported to great distances; for many facts could be given showing that soil almost everywhere is charged with seeds. Reflect for a moment on the millions of quails which annually cross the Mediterranean; and can we doubt that the earth adhering to their feet would sometimes include a few minute seeds? But I shall presently have to recur to this subject.

Almost every year, one or two land-birds are blown across the whole Atlantic Ocean, from North America to the western shores of Ireland and England; but seeds could be transported by these wanderers only by one means, namely, in dirt sticking to their feet, which is in itself a rare accident.

When a duck suddenly emerges from a pond covered with duck-weed, I have twice seen these little plants adhering to its back; and it has happened to me, in removing a little duck-weed from one aquarium to another, that I have quite unintentionally stocked the one with fresh-water shells from the other. But another agency is perhaps more effectual:

I suspended a duck's feet, which might represent those of a bird sleeping in a natural pond, in an aquarium, where many ova of fresh-water shells were hatching; and I found that numbers of the extremely minute and just hatched shells crawled on the feet, and clung to them so firmly that when taken out of the water they could not be jarred off, though at a somewhat more advanced age they would voluntarily drop off. These just hatched molluscs, though aquatic in their nature, survived on the duck's feet, in damp air, from twelve to twenty hours; and in this length of time a duck or heron might fly at least six or seven hundred miles, and would be sure to alight on a pool or rivulet, if blown across sea to an oceanic island or to any other distant point."
On the origin of Species Chapter 12 - Geographical Distribution