I'm all set to do the phage recombination assays I described earlier here and here. But the postdoc has a clever hypothesis that I hadn't thought of, that can be easily tested.
I set up our new/old incubators at 33°C and 41°C, and used them to assay the titers (plaque-forming units per ml; pfu/ml) of the phage lysates I made a couple of weeks ago. These temperatures turned out to be just right (this is a Goldilocks assay); the 41°C incubator wasn't too hot for the cells to grow and the 33°C incubator wasn't too cool for plaques to form. My lysates of the three temperature-sensitive mutant phages all had titers of 5-8 x 10^10 pfu/ml at 33°C and titers of less than 10^6 at 41°C. These high titers mean that it will be easy to set up mixed infections where most cells are infected by phage of two different genotypes, and the low background means I can sensitively detect recombinants.
My original plan was to screen all of our competence-gene knockout strains for the ability to promote phage recombination. This recombination has long been known to occur much more efficiently in competent cells and to not occur in the few competence mutants where it has been tested (crp, cya, sxy and rec2), so I've hypothesized that the ability to recombine phage DNA reflects a competence-induced change in DNA metabolism. This means that I can use phage recombination as a probe for the competence-induced changes in DNA metabolism.
BUT, this hypothesis fails to explain why phage recombination isn't induced by competence in the rec2 mutant. The defective Rec2 protein in rec2 mutants is thought to only block transfer of DNA from the periplasm to the cytoplasm. We know that competence-induced DNA uptake across the outer membrane is unaffected, and we expect that competence-induced cytoplasmic changes also occur normally. So why wouldn't phage recombination happen in these cells?
I had already considered one explanation. Phage recombination is known to need the Rec1 (RecA) pathway of homologous recombination. This pathway may be enhanced in competent cells, but not because the rec1 gene is part of the competence regulon. Rather, we think the Rec1 protein, like its RecA homolog in E. coli, is activated by the single stranded DNA that competent cells bring into the cytoplasm. DNA isn't deliberately added to cultures being tested for phage recombination, but I hypothesized that that normal cultures of competent cells might contain significant amounts of free DNA from cells that died during incubation in the starvation medium, and that uptake of this DNA might activate Rec1 recombination. I think I might even have done a test of this years ago, by adding DNase I to the starvation medium, but if so the results were inconclusive.
The postdoc has a better idea. He reminded me that phage lysates typically contain significant amounts of free phage DNA (DNA that had not been packaged into phage capsids before the cell lysed), and that competent cells can take up this DNA. So maybe the recombinant phage are produced not by recombination between the DNAs of two independently infecting phages replicating in the cytoplasm of a single cell, but by recombination between the DNA of a single infecting phage and DNA of a different phage that has been brought in by the competence machinery!
I don't know how much free phage DNA a lysate would contain, but I bet it's at least as much as the amount of DNA packaged in the phage particles. In recombination assays cells are typically incubated with both phages at a 'multiplicity of infection' greater than 1 to ensure that most cells are infected by both phages (that's why I need high-titer lysates), so these incubations also provide the cells with lots of free phage DNA that they may take up by the competence pathway. And we know that phage DNA is taken up efficiently because it contains lots of uptake sequence.
The easiest test of the postdoc's hypothesis is to incubate the mutant phage lysates with DNase I before mixing them with the cells. If he's right this should dramatically reduce phage recombination without affecting phage replication. This doesn't really distinguish between his hypothesis and mine, so a better test would be to purify DNA from the lysates and test recombination in infections where cells are incubated with a lysate of one mutant phage and DNA from a different mutant. I calculate that my lysates contain about 4 µg/ml of phage DNA in virions, so a simple DNA prep from a few ml should give me enough DNA for this test. Another test would be to precipitate out the intact phage from a lysate (I think this is easily done using polyethylene glycol (PEG)) and show that its titer is way down but its contribution to recombination is not.
I had thought of phage recombination as providing a way to probe the state of DNA metabolism, one independent of the usual assays of chromosomal transformation. But if phage recombination turns out to occur by uptake dependent recombination of translocated phage DNA (single-stranded) with replicating phage DNA, then it's just another transformation assay, differing only in the target being phage DNA rather than chromosomal DNA. It still might be a useful probe, but not in the way I was thinking.
End of summer &
13 hours ago in The Phytophactor