Classes have been over for a month and I have yet to do an experiment! For shame. But after going over everyone else's projects I think I have a clear idea of what I should be doing.
1. Measure starvation-induced competence for each of the competence-gene knockouts: This will be replicating measurements done by an undergraduate over the past 6 months. I'll just do each strain once, provided my results agree with hers. I'll also freeze some of the competent cells.
2. Assay phage recombination in the competent and non-competent mutant cells: Old experiments using temperature-sensitive mutants of the H. influenzae temperate phage HP1 showed that recombination between the ts loci is more frequent in competent cells and completely dependent on the host RecA pathway (undetectable in a rec1 mutant). Phage recombination is much more efficient in competent cells than in log-phase cells; this is true both when competence is induced by starvation of wildtype cells (in MIV medium) and when it is induced by the presence of the sxy1 hypercompetence mutation (in rich medium).
But phage recombination is unexpectedly blocked by a rec2 mutation. (The baroque history of rec2 is described in this post.) The rec2 mutant also does not have the peculiar DNA aberrations described in competent cells (more below about these). So I'm wondering if phage recombination can give us a transformation-independent window into DNA metabolism in competent cells. Hence my plan to test all the mutants.
The recombination assays are simple in principle: just infect cells with a mixture of two ts phages (ts1 and ts3), each unable to form plaques at 40 °C. The infections are done at the permissive temperature (34 °C), but the resulting lysates are titered at both 34 °C and 40 °C. The ratio of plaque-forming units (pfu) gives the recombination frequency. But in practice they're fussy. I need to have incubators at both temperatures, which will take some arranging as we only have one incubator at present, set at 37 C. I also need to fuss with getting the temperature control just right, as H. influenzae is pretty picky about this, and to make lysates of the mutant phages that have sufficiently high titers that I can do mixed infections (need more phage than cells). To make these lysates I need to grow up the lysogens carrying the wildtype and mutant phages, and so I first need to find out whether they survived the big freezer meltdown of 2005. I may also need to buy fresh mitomycin C to induce the phage - our stock has been stored in the freezer but it's more than 20 years old.
While I'm surveying competent wildtype and mutant cells I'm going to revisit an unsolved old problem - the unusual structural features of DNA in competent cells. This was first reported by Leclecrc and Setlow in 1975, based on studies using denaturing sucrose gradients which suggested that the chromosomal DNA of competent cells had single-stranded regions, such that when denatured the average fragment length is about 200 kb (if I've correctly converted 5x10^7 Daltons to kb). David McCarthy later used electron microscopy to examine competent cell DNA; he found single-strand gaps and 'tails' in DNA from competent cells. The gaps were not seen in a rec2 mutant, but the tails were seen in both rec1 and rec2 mutants. As a post-doc I tried to investigate this, using high molecular weight DNA purified from log-phase and competent wildtype cells. I incubated the DNAs with Klenow and 32P nucleotides to tag any gaps - this labelled the DNAs of competent cells and log-phase cells identically. DNA from stationary-phase cells was labelled less, presumably because of the absence of replication forks. I also tried digesting the DNAs with nuclease S1, which preferentially cuts single-stranded DNA, but I had very odd results, as the competent-cell DNA migrated very strangely after incubation with the special buffer used for S1 nuclease (NaOAc ph 4.5 10 mM Zinc, 1.5 M NaCl). It's this strangeness that I now want to revisit, so I'll just make DNA from competent and log-phase cells the usual way, incubate it with this and other buffers, and run it in gels with and without restriction digestion.
Welcome to the 4th Reich part 1.
6 hours ago in Angry by Choice