I'm using fusions of lacZ to the comA and rec2 competence genes to find out whether the HI0659 mutation acts by blocking competence induction. The first step was to put the HI0659 mutation (strain RR3112, HI0659::spc) into strains carrying these fusions. That was easy, because the fusion strains are still transformable (the fusions were introduced as duplications of the chromosomal comA and rec2 genes rather than as replacements), and because I can select for the HI0659 mutation using spectinomycin.
We has two versions of each fusion strain in the freezer - the original strains sent to us by their creator, and derivatives we'd made by transforming the fusions into our standard strain KW20. I decided to start by using them both, in case anything wonky turned up.
So I made the strains competent and transformed them with RR3112 DNA and, as a control, with our standard MAP7 DNA. Both transformations worked fine, with transformation frequencies between 10^-3 and 10^-2. I streaked two colonies from each RR3112 transformation onto chloramphenicol plates to make sure they still had the fusion - only one didn't. The next steps are to freeze these now strains (in case we want to do more with them), and to make them competent by incubation in MIV starvation medium. I'll then test the competent cells for transformation (should be negative) and for expression of the lacZ fusions.
But first I needed to check that competence induction did induce the fusions on the parent strains. When I had made these competent (for the RR3112 transformations) I had frozen aliquots of log-phase and competent cells, so I thawed them out and did beta-galactosidase assays on them. My first set of assays were a complete failure (no yellow colour even after 18 hours!), because I'd used 10% SDS rather then 0.1%, but the second set worked great, with bright yellow colour after 20 minutes.
Here's the graph:
I forgot to label the XY-axis - it's the OD420 reading, indicating the level of expression of the fusion. (I didn't bother to convert these numbers into Miller units.) Three of the strains have almost no fusion expression in log phase and high expression after competence induction, which is what we expect of strains with normal competence regulation. But the fourth strain (878) has high expression in log phase, because it also carries a mutation (murE749) that causes the competence genes to be highly induced even in log phase, giving a 'hypercompetent' phenotype. I'll include the HI0659 derivative of this strain in my assays as a control.