POLYAMINES, RNA SYNTHESIS, AND STREPTOMYCIN LETHALITY IN A RELAXED MUTANT OF E. coli STRAIN 15 TAU

Abstract

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in cultures of TAU lacking arginine;' RNA accumulated in the presence of spermidine is predominantly ribosomal RNA (unpublished data).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

Interrelation Between Guanosine Tetraphosphate Accumulation, Ribonucleic Acid Synthesis, and Streptomycin Lethality in Escherichia coli CP78

“…Later studies by Freda and Cohen (7) showed that the synthesis of 16S ribosomal RNA is specifically stimulated by streptomycin and it was suggested that the synthesis of this class of RNA is in some way related to cell death. Consistent with this hypothesis was the observation that the isogenic relaxed mutant, 15 T-A-U-(rel-), which synthesizes ribosomal RNA actively in the absence of arginine, was killed much more rapidly by streptomycin (3).…”

Regulation of Macromolecular Synthesis by Putrescine in a Conditional Escherichia coli Putrescine Auxotroph

“…In the experiment illustrated in Fig. 2A, the concentrations of thymine were 0.10 MCi/ml and 4 Mg/ml. To assist thymine uptake, deoxyguanosine (500 gg/ml) was also added to the medium.…”

Biosynthesis and Metabolism of 1,4‐diaminobutane, Spermidine, Spermine, and Related Amines

“…Earlier work (77,97,410) described the occurrence of monoacetyl-1,4diaminobutane and of monoacetylspermidine in E. coli. * However, more recent studies have shown that E. coli, growing in a glucose-salts medium, does not contain any acetylated polyamines (405,406).…”