Wild-type Escherichia coli cells grow normally in 95% O2͞5% CO2. In contrast, cells that cannot make polyamines because of mutations in the biosynthetic pathway are rapidly killed by incubation in 95% O2͞5% CO2. Addition of polyamines prevents the toxic effect of oxygen, permitting cell survival and optimal growth. Oxygen toxicity can also be prevented if the growth medium contains an amino acid mixture or if the polyamine-deficient cells contain a manganese-superoxide dismutase (Mn-SOD) plasmid. Partial protection is afforded by the addition of 0.4 M sucrose or 0.4 M sorbitol to the growth medium. We also report that concentrations of H2O2 that are nontoxic to wild-type cells or to mutant cells pretreated with polyamines kill polyamine-deficient cells. These results show that polyamines are important in protecting cells from the toxic effects of oxygen. It is well known from the classic studies of Fridovich and his group (1-5) and others (6-8) that Escherichia coli cells, grown in air, have mechanisms for the protection of the cells from the toxic effects of superoxide and of oxygen radicals. Of particular importance is the protective effect exerted by the superoxide dismutase that is present in these cells. It has also been observed that E. coli cells can grow at a normal rate in an atmosphere of increased oxygen, presumably by operation of these protective mechanisms (7).Polyamines, the ubiquitous polycationic compounds, are associated with a variety of biological processes, such as nucleic acid biosynthesis, cell growth, and differentiation (9, 10). In this paper, we present data showing that another important factor in the protection of E. coli cells from the toxic effects of oxygen is the intracellular level of polyamines. We have found that polyamine-deficient cells are rapidly killed by incubation in oxygen, even though, as we previously reported, when incubated in air, these same cells are not killed and grow indefinitely (albeit at a somewhat reduced growth rate; ref. 11). Materials and MethodsThe strains used are listed in Table 1. All strains were maintained on LB plates. All incubations were at 37°C with vigorous shaking. The various mutants were grown on a purified polyamine-free Vogel-Bonner (VB) medium ref. 16) or M63 medium (Fig. 5; ref. 7) [supplemented with glucose and the auxotrophic requirements for EWH319 and HT306 (thiamin, pantothenate, proline, and threonine)] for at least 20-25 generations to deplete any polyamines remaining from the original LB medium. The cultures then were grown overnight in limiting glucose (0.025%) to an OD 600 of 0.1-0.2. Additional glucose (0.4%) was then added, plus polyamines where indicated, and the cultures were incubated for another 2 h. The cultures were then diluted to an OD 600 of 0.001 into the same medium with and without polyamines. † In some of the experiments an amino acid mixture (Fig. 3A), sucrose or sorbitol (Fig. 3B) was added at this point. For experiments presented in Fig. 5 (with pDT1.5, sodA plasmid), the cultures were grown in M63 medium...

Spermidine and its derivative, hypusinated eIF5A, are essential for the growth of Saccharomyces cerevisiae. Very low concentrations of spermidine (10 ؊8 M) are sufficient for the growth of S. cerevisiae polyamine auxotrophs (spe1⌬, spe2⌬, and spe3⌬). Under these conditions, even though the growth rate is near normal, the internal concentration of spermidine is <0.2% of the spermidine concentration present in wild-type cells. When spe2⌬ cells are grown with low concentrations of spermidine, there is a large decrease in the amount of hypusinated eukaryotic initiation factor 5A (eIF5A) (1/20 of normal), even though there is no change in the amount of total (modified plus unmodified) eIF5A. It is striking that, as intracellular spermidine becomes limiting, an increasing portion of it (up to 54%) is used for the hypusine modification of eIF5A. These data indicate that hypusine modification of eIF5A is a most important function for spermidine in supporting the growth of S. cerevisiae polyamine auxotrophs.eIF5A ͉ protein synthesis ͉ yeast P olyamines (putrescine, spermidine, and spermine) (Scheme 1) are present in micromolar-to-millimolar concentrations in prokaryotic and eukaryotic cells and play important roles in cell growth and development (1, 2). Intracellular levels of polyamines are regulated at various steps, including synthesis, degradation, uptake, and excretion, and cells have developed intricate mechanisms to ensure tight regulation of intracellular polyamine pools (3, 4). We have found that, of the three amines, spermidine is most important for cell growth, and near-normal growth is obtained in mutant cultures containing extremely low concentrations of spermidine (5-7).One of the important functions of spermidine is its role as a substrate for the hypusine modification of the putative eukaryotic translation initiation factor 5A (eIF5A) (Scheme 1). eIF5A is a highly conserved and essential protein present in all organisms from archaebacteria to mammals (8-10), and the hypusine/ deoxyhypusine modification of eIF5A is essential in all eukaryotic cells (11). Hypusine was first isolated from a brain extract by Shiba et al. (12), and later Folk and colleagues (13)(14)(15) showed that the eIF5A precursor undergoes a unique posttranslational modification to form the hypusine residue. This modification occurs exclusively in this protein and proceeds by two consecutive steps. First, precursor eIF5A is modified by deoxyhypusine synthase, where the aminobutyl group of spermidine is attached to a specific lysine residue, followed by hydroxylation of the intermediate by deoxyhypusine hydroxylase to form the active hypusinated eIF5A (16).Hypusine modification by spermidine is essential for growth and protein synthesis (8, 10, 11) and in our current studies, we show that when a polyamine auxotroph is grown in the presence of very limiting concentrations of spermidine, most of the spermidine is used for the modification of eIF5A. Further support for the concept that this modification of eIF5A is the major function of sperm...

In our earlier work we showed that either spermidine or spermine could support the growth of spe2⌬ or spe3⌬ polyamine-requiring mutants, but it was unclear whether the cells had a specific requirement for either of these amines. In the current work, we demonstrate that spermidine is specifically required for the growth of Saccharomyces cerevisiae. We were able to show this specificity by using a spe3⌬ fms1⌬ mutant that lacked both spermidine synthase and the FMS1-encoded amine oxidase that oxidizes spermine to spermidine. The polyamine requirement for the growth of this double mutant could only be satisfied by spermidine; i.e., spermine was not effective because it cannot be oxidized to spermidine in the absence of the FMS1 gene. We also showed that at least one of the reasons for the absolute requirement for spermidine for growth is the specificity of its function as a necessary substrate for the hypusine modification of eIF5A. Spermine itself cannot be used for the hypusine modification, unless it is oxidized to spermidine by the Fms1 amine oxidase. We have quantified the conversion of spermine in vivo and have shown that this conversion is markedly increased in a strain overexpressing the Fms1 protein. We have also shown this conversion in enzymatic studies by using the purified amine oxidase from yeast.eIF5A ͉ polyamine ͉ polyamine oxidase P olyamines, such as putrescine, spermidine, and spermine, are ubiquitous cellular polycationic compounds. Their essential nature has been demonstrated by many studies with inhibitors and, in particular, with mutants in the biosynthetic pathway (1-3).In many studies some of the phenotypic effects of spermidine and spermine have been interchangeable. This has also been true of our recent studies with Saccharomyces cerevisiae. Thus, strains unable to synthesize spermidine because of deletions (spe2⌬ or spe3⌬) in the biosynthetic pathway † did not grow in amine-free medium unless spermidine or spermine was added to the growth medium (4, 5). However, because either spermidine or spermine permitted growth, it was unclear whether the cells had any specific requirement for spermidine that could not be satisfied by spermine. In the present work we show that S. cerevisiae cells do indeed have an absolute requirement for spermidine and that this requirement cannot be met by spermine unless the spermine is first oxidized to spermidine by an amine oxidase.We also show that at least one of the reasons for the absolute requirement for spermidine (and not spermine) for growth is the specificity of its function as a necessary substrate for the hypusine modification of eIF5A. eIF5A is a eukaryote protein synthesis initiation factor that is essential for growth (6-10) and may be involved in mRNA metabolism, translation, and ribosome biogenesis (11,12). In many cell types hypusine modification has been shown to occur by the transfer of the 4-aminobutyl moiety of spermidine to the -amino group of a specific lysine residue of precursor eIF5A (13,14). Studies with yeast mutants have shown that ...