Stimulation of phiX174 production in mitomycin C-treated Escherichia coli cells by caffeine.
چکیده
After pretreatment of an Escherichia co/i C culture with mitomycin C, the deoxyribonucleic acid (DNA) synthesis of the bacteria, as well as that of infecting phage 4X174, is very largely inhibited. Although the bacterial DNA synthesis is similarly inhibited by mitomycin C in uvrmutants (P. Howard-Flanders, R. P. Boyce, and L. Theriot, Genetics 53:1119, 1966), phage DNA synthesis and progeny formation take place in a normal manner (B. H. Lindqvist and R. L. Sinsheimer, J. Mol. Biol. 30:69, 1967). The reason for this behavior of the uvrcells is not understood. Since it is known that uvr+ cells do not reactivate double-stranded ultravioletdamaged phage DNA in the presence of adequate concentrations of caffeine (W. Sauerbier, Biochem. Biophys. Res. Commun. 14:340, 1964), it was of interest to test the ability of mitomycin C-treated uvr+ cells to produce phage under these conditions. A thymine-requiring mutant ClT (isolated by M. Azegami) was used in the experiments. The CTbacteria were grown with aeration at 37 C in a modified TPG medium (R. L. Sinsheimer et al., J. Mol. Biol. 4:142, 1962; 1 g/liter of KH2PO4, 0.147 g/liter of CaCI2-2H2O _ 10-3 M), SUpplemented with 2.7 g/liter of a special mixture of the 20 natural L-amino acids (Nutritional Biochemicals Corp., Cleveland, Ohio) and 0.01 g/liter of thymine. At a titer of approximately 6 x 107 colony formers per ml, the bacteria were concentrated fivefold in the same medium but with only one-fifth of the thymine concentration. The cells were treated with mitomycin C in a dim-yellow light without aeration for 15 min at 50 ,ug/ml and at 37 C. The caffeine concentrations employed were 1, 3, and 10 mg/ml; normally the caffeine treatment began with the addition of the mitomycin. Mitomycin was then removed by centrifugation, and phage was added (m 5) to the resuspended cells. Phage adsorption took place (in the presence of caffeine) during 7.5 or 10 min, whereupon the bacteria were diluted 1 :100 in the same medium and incubated until lysis (1 hr after addition of wild-type phage) or artificial opening (at 40 or 60 min after addition of the lysis-defective muTABLE 1. Relative phage yield