I finally found some time to do more phage recombination work. I still need to repeat the experiment showing that DNase I pretreatment of lysates reduces phage recombination but not phage titer) and that protease pretreatment reduces phage titer but not phage recombination. But this experiment was instead testing whether phage recombination is affected by mutations that eliminate DNA uptake or reduce chromosomal transformation.
Each test involves infecting cells with amixture of two temperature-sensitive phage mutants (ts1 and ts3), and plating the progeny phage at the restrictive temperature of 41 °C. I tested two uptake-null mutants, pilA and rec2. rec2 mutants are known to be defective for phage recombination; pilA has never been tested but I expected the same phenotype, based on the hypothesis that phage recombination depends on uptake of phage DNA by the competence machinery. I also tested knockouts of three cytoplasmic-protein genes in the competence regulon, dprA, comM, and radC.
As positive controls I tested phage recombination in competent wildtype cells, and phage production using wildtype phage. As negative controls I tested phage recombination in log-phase wildtype cells (no recombination expected), and infection of wildtype cells with each phage mutant singly (no plaques expected at the restrictive temperature).
The positive control infection gave a recombinant frequency of 5 x 10^-3, as expected, and the single-infection controls worked well - no plaques at 41 °C, giving revertant frequencies of <3 x 10^-6 (ts1) and 3 x 10^-5 (ts3). But the negative phage-recombination control (log-phase cells) and the known recombination-negative mutant (rec2) both gave way more plaques at 41°C than expected (frequencies of 2 x 10^-4 and 9 x 10^-5 respectively).
Three of the previously untested mutants had recombination frequencies similar to the negative controls (pilA: 1.6 x 10^-4; dprA: 1.3 x 10^-4; comM: 2.7 x 10^-4), and the radC mutant, which has normal frequency of chromosomal transformation, had a near-normal frequency of phage recombination (1.2 x 10^-3). But these values are not very useful because of the high background. Need to do it again.
Welcome to the 4th Reich part 1.
16 hours ago in Angry by Choice