Tuesday, March 18, 2008

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

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.

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