We have studied the biosynthesis of polyamines during the multiplication of the RNA bacteriophage R17. R17-sensitive strains of Escherichia coli were derived from the stringent CP78 and the relaxed mutant derivative CP79. The cells were infected with R17 in the presence or absence of arginine, a required amino acid, and both the RNA and polyamine contents of the bacteria were determined before and after the infection. The uninfected CP79 rel derivative accumulated RNA and spermidine in the absence of arginine, unlike the stringent organism that accumulated neither under these conditions. After R17 infection, the stringent strain accumulated RNA and spermidine in the presence or absence of arginine. The data indicate a close correlation between the synthesis of RNA and spermidine, suggesting a significant role for this polyamine in the multiplication of phage R17.
The rate of protein synthesis by Escherichia coli markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection. Casamino Acids to a final concentration of 0.2%c. TY broth contained 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter. Growth and infection of bacteria. Cells of E. coli B were grown at 37 C in an Eberbach reciprocal shaker. Growth was measured in a Klett colorimeter, and the Klett reading had been calibrated to the number of colony formers in the culture media. The mass doubling times were 50, 50, and 30 min, in TG medium, TGA medium, and TY broth, respectively. Cells were grown to a densitv of I to 5 X 108 cells/ml and were 713
Bacteriophage R17 and its RNA were found to contain significant amounts of spermidine but not of putrescine. When isolated at 0.01 M KCl, up to 1,000 molecules of spermidine were associated with the virion. The phage RNA isolated with phenol plus sodium lauryl sulfate contained approximately 70 to 90 molecules of spermidine. The association appeared to be ionic because the bound spermidine could be dissociated by KCI, MgCl2, or both. Effects of polyamines on in vitro translation were studied using both poly(U) and phage R17-RNA as mRNA. Addition of spermidine to the system at suboptimal concentrations of Mg2+ resulted in marked stimulations of the rate of protein synthesis. Putrescine alone had no effect but stimulated the incorporation in the presence of suboptimal concentrations of spermidine plus Mg2+. The isolated amino acid-incorporating system contained suboptimal soluble and bound polyamines. A comparison of incorporation was made in this system using R17-RNA with and without bound spermidine. No effects of these bound cations were detected on the rate or extent of incorporation of valine. The ratio of incorporation of histidine (present in noncoat proteins) to valine (total protein) revealed little difference as a function of cation in the system or as a function of the spermidine present in R17-RNA.
A previous paper (1973) indicated that infection with bacteriophage R17 permits the synthesis of RNA and spermidine in Escherichia coli CP78 in the absence of the exogenous essential amino acid, arginine. We have now isolated RNA formed under such conditions and analyzed the newly synthesized species by agarose-acrylamide electrophoresis. It has been shown that infection of the stringent cells irn the absence of exogenous arginine resulted in a marked incorporation of uracil into rRNA, as well as into R17 RNA. It was shown that, although the organism was nonauxotrophic for uracil, addition of ["4C]uracil
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