As I posted a few days ago, I planned to introduce a sxy-expression plasmid into my cells with CRP-S promoter fusions to lacZ, to see if the CRP-S promoters are indeed induced by Sxy. I did that yesterday, and I just finished calculating the amounts of beta-galactosidase activity these cells produced with and without induction of sxy expression.
Results: Of the three fusions I tested (all that I have), one produced almost 100-fold more beta-galactosidase when Sxy was present, one produced only slightly more, and one produced even less! The exclamation mark is because I expected them all to respond similarly.
The fusion that was induced by Sxy is to the ppdA promoter (really the promoter of a 4-gene operon). The H. influenzae homologs (not pilB but comNOPQ) are in a similar operon, which is very strongly induced in competent cells and strongly dependent on CRP and Sxy. The ComN and ComO proteins are known to be required for competence. This fusion is carried on a plasmid. The 100-fold induction when Sxy is overexpressed is what I was hoping to see. This means that the low expression when Sxy is not induced is indeed baseline, so I'm comfortable with this baseline expression not being dependent on Sxy or CRP.
The results with the other fusions are surprising. The one that produced only slightly more is a plasmid-borne fusion of lacZ to the hofM promoter (really the promoter of the hofMNOPQ operon). This is homologous to H. influenzae's comABCDE operon, and like comNOPQ it is required for competence and needs CRP and Sxy for induction. Its CRP-S promoter element doesn't look significantly worse than that of ppdA, but it was induced only three-fold by Sxy overexpression.
The fusion that produced even less beta-galactosidase when Sxy was induced is to the ppdD promoter; it's integrated into the chromosome rather than being carried on a plasmid. This is the most surprising result, as the grad-student-now-postdoc-in-waiting ('gsnpiw') used quantitative PCR to show that ppdD mRNA is induced about 100-fold by overexpression of Sxy from the same plasmid I just used. Induction of Sxy in the fusion-carrying cells not only failed to induce the ppdD::lacZ fusion, it actually made these cells quite sick; the cells with IPTG grew to less than half the density of the uninduced cells, and produced only about half as much beta-galactosidase per cell. Perhaps the chromosomal insertion in these cells has somehow made them sensitive to transcriptional activation of the ppdA gene.
Anyway, the good news is that I can use the ppdA fusion as an indicator of Sxy expression, in my search for conditions that induce Sxy and thus induce expression of CRP-S genes. I'll set aside (for now) the questions about why the other fusions don't behave similarly.
(Sorry I've been too lazy to include figures in my recent blog posts; I promise to improve.)
If at first you don't succeed, put it all back they way it was and keep lumbering along
11 hours ago in The Phytophactor