So I did some beta-galactosidase assays on the strain with ppdD fused to lacZ. The immediate goal was to characterize baseline transcription of the ppdD gene, with the longer goal of finding ways to turn it up by inducing expression of sxy.
The researcher who kindly sent us the strain with this fusion said that its colonies are pale blue on plates with the beta-gal indicator X-gal, but for me they were quite a strong blue. So I wasn't too surprised that my assays showed moderate beta-gal activity in all the conditions I tested. I tested cells in exponential growth and after overnight culture in LB, in LB+glucose (which should prevent production of cAMP and thus expression of the ppdD gene's CRP-S-regulated promoter, and of cells in LB+glycerol, which should allow cAMP production like plain LB but provide the same amount of extra carbon source that glucose does. I also tested cells transferred from log-phase growth in LB+glucose to minimal salts with added amino acids ("M9+caa"); a treatment that might roughly approximate the competence-inducing effect of transferring H. influenzae cells from sBHI to MIV. None of these treatments made much difference; all the samples produced between 150 and 500 units of enzyme activity per ml of cells.
So the next test is to find out whether the transcription of ppdD depends on Sxy. If it does, then this suggests that Sxy is being produced at least a bit under the standard conditions I tested. If not, the expression is genuinely baseline, and maybe I should test other genes as indicators of sxy expression.
To do this test I need to introduce our sxy knockout into the ppdD::lacZ fusion strain. So tomorrow I'll go searching for the needed P1 lysate. The colleague who has it has unfortunately gone off to Europe for a couple of months, but he tells me that one of the people in his lab can help me find it.
11 hours ago in Variety of Life