I've been thinking about the phenotypes of our confirmed purR::kan mutant and of the other pseudo-purR mutant (I'll call it 'purR*'). And I'm wondering whether the confirmed mutant might have some residual purR function, and the purR* mutant might be the true loss-of-function mutant.
We rather naively assumed that a cassette insertion in the middle of the purR gene would knock out its function. But the PurR protein, like its close relative LacR, has two domains, a small N-terminal domain that binds its recognition site and a larger C-terminal domain that interacts with the corepressors guanine and hypoxanthine and with the C-terminal domain of a second PurR protein, forming the active dimers. And our cassette insertion's central position means that it knocks out the domain responsible for corepressor binding and dimerization, but leaves the DNA binding domain intact.
We don't know whether monomers of the PurR DNA-binding domain can actually bind DNA, but in E. coli those of LacR can (weakly). Thus our supposed knockout may not allow maximal transcription of the genes PurR normally represses. Not only would the genes still be a bit repressed under what are normally repressing conditions, but they would remain a bit repressed even under what would otherwise be maximally inducing conditions (because the corepressor binding sites are in the other domain). Furthermore, in E. coli the PurR protein represses transcription of the purR gene (it autoregulates) - if this occurs in H. influenzae then our supposed purR knockout might actually be overproducing its DNA-binding domain!
If the purR mutant isn't a true knockout, perhaps the purR* mutant is. Because this strain was created by transforming wildtype cells with what was supposed to be purR::kan DNA, and because its phenotype is a more extreme version of the purR::kan strain's mild late-log competence defect, the purR gene is the strongest candidate location for whatever differentiates purR* from wildtype.
We need to get this sorted out quickly, because if our purR::kan mutant isn't a knockout we shouldn't use it to analyze gene regulation. Today I did another time course of competence in sBHI, directly comparing the wildtype, purR and purR* strains, to make sure our conclusions about the phenotypic differences are correct.
The RA is going to quickly get the purR allele of the purR* strain sequenced. I'm hoping that we'll find that it has a frameshift or other mutation that inactivates both domains of PurR, creating a true knockout. We'll still also want to find out where its kan cassette is - we don't have enough genetic tools to locate it genetically, so inverse PCR will probably be best.
Regardless of what the purR* sequencing shows, we should probably make a new purR::kan mutation, one that deletes most of the gene so we can be sure there's no residual activity.
Welcome to the 4th Reich part 1.
16 hours ago in Angry by Choice