Polyamine Stimulation of Nucleic Acid Synthesis in an Uninfected and Phage-Infected Polyamine Auxotroph of Escherichia coli K12

Abstract

The addition of arginine to cultures of Escherichia coli K12 deficient in agmatine ureohydrolase (EC 3.5.3 Two constitutive pathways function in the synthesis of putrescine in Escherichia coli (1, 2). In the E. coli K12 mutant deficient in agmatine ureohydrolase (EC 3.5.3.7) (3), the conversion of agmatine to putrescine is blocked. The addition of arginine prevents putrescine formation in this mutant by inhibition of the synthesis of ornithine from glutamate. Such a polyamine auxotroph has permitted us to investigate the dependence on polyamines of RNA and DNA synthesis in uninfected cells and the possible requirement for polyamines for DNA synthesis in phage-infected cells.In 1957, Hershey (4) described two low molecular weight ninhydrin-positive compounds in purified T-even phage preparations; these amines were also demonstrated to be present in the uninfected host. These compounds were shown to be derived from arginine, and were concomitantly injected with phage DNA upon infection; they were identified as putrescine and spermidine by Ames and coworkers (5, 6), who showed that the polyamine content of the T-even phage could neutralize 40-50% of the DNA phosphates. Cohen and Raina (7) have demonstrated that the net synthesis of polyamines after infection parallels DNA synthesis. The MATERIALS AND METHODSSome properties of E. coli K12 (MA159) (His-, Leu-, Thr-) defective in agmatine ureohydrolase activity have been described (3). The characterization of bacteria bearing this block has been described (3, 9). The mutant that was used in this study, MA159, was kindly given to us by Dr. W. Maas. The organism is derived from a thy-derivative (MA145) of MA135 (3) by transduction into a ser A strain, PA260. In this study, two methods of polyamine depletion were used. In method 1 (J. Poindexter, personal communication), an overnight broth culture is chilled 5 hr at 4°C and diluted 1: 20 in AFA medium (AF medium, see ref. 3, supplemented with 100 ,ug of arginine/ml). 0.1-ml aliquots are plated on AFA agar plates, and after overnight growth at 37°C, bacteria are scraped from the agar surface with small aliquots of AFA medium. These harvested bacteria are then centrifuged at 4080 X g for 15 min, and resuspended in Davis-Mingioli medium (9) without glucose. After an additional wash, aliquots of the suspension are added to various AF media containing the designated supplements.In method 2, an overnight broth culture is chilled 5 hr at 4°C and diluted 1:40 in AFA medium (final concentration of arginine = 100 ,g/ml). Overnight growth at 37°C results in polyamine-depleted cells. These are centrifuged and washed in Davis-Mingioli salts medium without glucose. Aliquots of a 4-fold concentrated suspension are added to AF media containing the designated supplements.Lysates of T4r+D were prepared by infection of E. coli B at a multiplicity of infection (MOI) of 0.1 and incubation overnight. Purified phage stocks were obtained by two cycles of low (4080 X g)-and high (27,300 X g)-speed centrifugation of overnight brot...

Growth and Macromolecular Composition of a Mutant of Escherichia coli During Polyamine Limitation

“…However, a low level (approximately 4 nmoles/mg of protein) of a compound which behaved like cadaverine on column chromatography (18,20) and on thin-layer chromatography (18) was observed. This observation is in agreement with the results of Dion and Cohen (8). In strain CJ352, grown in amino acid-supplemented medium with putrescine supplementation, it appears that most of the putrescine taken up was converted to spermidine as reflected by the fact that the intracellular putrescine levels were still below the limits of detection.…”

Isolation, characterization, and mapping of Escherichia coli mutants blocked in the synthesis of ornithine decarboxylase

“…Lysine decarboxylase can be induced by growing cells in a high concentration of lysine at low pH. In addition, cadaverine may appear as a constituent of the polyamine pool in the absence of lysine supplementation and at the physiological pH after polyamine depletion (2). Cadaverine may substitute for putrescine as an alternative substrate for aminopropyl transfer and does have growth-stimulating effects in these strains.…”

“…Polyamine pool measurements. The fluorimetric technique used for measurement of the polyamine pool (2) was modified slightly from that of Leifer (Ph.D. thesis).…”

The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

“…Moreover, the presence of cadaverine and APC in cell extract of P. furiosus is unusual and indicates that cadaverine can act as a propylamine acceptor from decarboxy-AdoMet in the synthesis of APC. It is interesting to note that APC was identified in a polyamine-requiring mutant of E. coli, grown in the absence of polyamines (14). Higher homologs of cadaverine, such as APC, were also synthesized as a compensatory polyamine by E. coli polyamine-requiring mutants (24).…”