The cellular accumulations of polyamines and ribonucleic acid (RNA) were compared in the polyauxotrophic mutants of Escherichia coli strain 15 TAU and E. coli K-12 RCrel met-leu-. Putrescine, spermidine, and their monoacetyl derivatives were the main polyamines in both strains, when grown in glucose-mineral medium. No significant degradation of either "4C-putrescine or '4C-spermidine was found in growing cultures of strain 15 TAU, which requires thymine, arginine, and uracil for growth. Experiments with this organism showed that in a variety of different incubation conditions, which included normal growth, amino acid starvation, inhibition by chloramphenicol or streptomycin, or thymine deprivation, a close correlation was seen between the intracellular accumulation of unconjugated spermidine and RNA. In the presence of arginine, the antibiotics stimulated the production of putrescine and spermidine per unit of bacterial mass. Deprivation of arginine also resulted in an increase in the production of putrescine per unit of bacterial mass, most of which was excreted into the growth medium. However, in this system the antibiotics reduced the synthesis of putrescine. Furthermore, streptomycin caused a rapid loss of cellular putrescine into the medium. The latter effect was not seen in anaerobic conditions or in a streptomycin-resistant mutant of 15 TAU. Methionine added to the growth medium of growing TAU not only markedly increased the total production of spermidine, but also increased both the intracellular concentration of spermidine and the accumulation of RNA. Exogenous spermidine extensively relaxed RNA synthesis in amino acid-starved cultures of 15 TAU. Analysis in sucrose density gradients showed that the RNA accumulated in the presence of spermidine was ribosomal RNA. Cells of E. coli K-12 RCrel met-leu-, grown in a complete medium, had approximately the same ratio of free spermidine to RNA as did strain 15 TAU. However, the relaxed strain showed a much lower ratio of putrescine to spermidine than the stringent 15 TAU. Omission of methionine stopped spermidine synthesis and markedly increased both the intracellular accumulation and the total production of putrescine. It seems that a high intracellular level of spermidine acts as a feedback inhibitor in the biosynthesis of putrescine in this strain. The hypothesis that the intracellular concentration of polyamines may participate in the control of the synthesis of ribosomal RNA in bacteria is discussed. The wide distribution of natural polyamines in Tabor and Tabor (25). Models have been deliving organisms has raised the problem of their veloped (11) to explain the binding of spermidine physiological function. It has long been known and spermine to deoxyribonucleic acid (DNA). that polyamines are essential growth factors for The strong affinity of polyamines to nucleic acids several microorganisms. It has been shown that and their occurrence in ribosomal fractions (3, 19, polyamines are able to stabilize membrane struc-27) suggest that polyamines serve as cati...

We have recently described the synthesis of nucleic acid and of the polyamines putrescine and spermidinel" 2 in a stringent derivative of Escherichia coli strain 15 requiring thymine, arginine, and uracil (TAU st).3 4 The elimination of arginine from the medium not only results in the inhibition of RNA synthesis by about 90 per cent, thereby defining "stringency," but also prevents the cellular accumulation of spermidine.' The relaxation of ribosomal RNA synthesis in TAU st is effected by streptomycill2 which concomitantly kills the cells, expels cellular putrescine, and causes an increase of cellular spermidine. l We have suggested that the lethality of streptomycin requires the synthesis of ribosomal RNA.5Exogenous spermidine itself relaxes RNA synthesis extensively in cultures of TAU lacking arginine;' RNA accumulated in the presence of spermidine is predominantly ribosomal RNA (unpublished data). It appears then that the synthesis of ribosomal RNA, but not of DNA,2 in stringent E. coli is correlated with both the absolute level of cellular spermidine and the relative level of spermidine to total polyamine. Such an effect on RNA synthesis in vivo appears at least superficially similar to the initiation of RNA synthesis in vitro by addition of spermidine to a stalled RNA polymerase, as summarized elsewhere.2 As discussed earlier,2 an isogenic pair of relaxed and stringent auxotrophic strains might provide a useful system for the study of the relation of polyamines to RNA synthesis. A relaxed derivative of E. coli strain TAU st has recently become available. Our comparative analyses of the relaxed (TAU rel) and the stringent (TAU st) organisms have now revealed that TAU rel makes far more spermidine than does TAU st. Furthermore, spermidine synthesis in TAU rel is independent of RNA accumulation. During amino acid starvation, an accumulation of intracellular spermidine in TAU rel sharply inhibits putrescine biosynthesis. It appears also that relaxed strains are hypersensitive to streptomycin in amino acid deficiency, such sensitivity being apparently dependent on the production of ribosomal RNA. We also have observed that the addition of methionine, the spermidine precursor, to TAU st also increases the effect of streptomycin in both stimulating RNA synthesis and the lethality of the antibiotic.Materials and Methods.-Spermidine trihydrochloride, uracil-2-C14, and thymine-2-Cl4 were purchased from the California Corporation for Biochemical Research. The spermidine trihydrochloride is recorded as having a melting point of 261-2620, N = 16.30 per cent; we have found by chromatography and paper electrophoresis that this material contains less than 1 per centA of any ninhydrinreactive impurity. Putrescine-1,4-Cl4 -2 HCl was obtained from New England Nuclear Corporation. Streptomycin sulfate was obtained from the Nutritional Biochemicals Corporation. 721

Philadelphia), MEEK1 JANSEN, AND SEYMOUR S. COHEN. Polymer synthesis in killed bacteria: lethality of 2',3'-dideoxyadenosine. J. Bacteriol. 92:565-574. 1966.-We studied the metabolic capabilities of cells that had lost the ability to multiply under a variety of lethal treatments. Cultures of a polyauxotrophic mutant of Escherichia coli strain 15 requiring thymine, arginine, uracil, and adenine for growth were killed to a few per cent survivors by several different methods. These treatments included streptomycin, thymineless death, D-arabinosyladenine (ara-A), and 2',3'-dideoxyadenosine (DDA). The killed cells were washed and supplied with complete media, and were compared with control cells with respect to the ability to incorporate thymine, arginine, and uracil. Cells killed with streptomycin in the absence of thymine were only partially inhibited in deoxyribonucleic acid (DNA) synthesis; they were markedly inhibited in synthesis of ribonucleic acid (RNA) and protein. Cells that had suffered thymineless death were essentially uninhibited in DNA synthesis, partially so in RNA synthesis, but extensively inhibited in protein synthesis. Killing by ara-A did not prevent DNA synthesis, but markedly inhibited RNA and protein synthesis. The lethality of DDA was studied in the presence of exogenous adenosine; lethality was partially prevented by deoxyadenosine. Dideoxyadenosine was similar to ara-A and thymineless death in killing in a pattern in which RNA and protein synthesis continued while DNA synthesis was inhibited. Cells killed by DDA, however, were markedly inhibited in subsequent thymine incorporation, unlike cells killed by the other methods. In addition, at this time, the DDA-killed cells were more inhibited in incorporation of arginine than of uracil. DDA also potentiated thymineless death; when cells were killed rapidly by the combined treatment, only the ability to synthesize DNA was lost irreversibly. This agent (DDA) may permit the detailed study in E. coli of the relation of DNA synthesis to numerous phenomena, such as genetic recombination, sequential transcription, and the number and distribution of chromosome breaks.