Herbert Tabor

Polyamines protect Escherichia coli cells from the toxic effect of oxygen

Wild-type Escherichia coli cells grow normally in 95% O2/5% CO2. In contrast, cells that cannot make polyamines because of mutations in the biosynthetic pathway are rapidly killed by incubation in 95% O2/5% 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.

Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine

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 (spe1Δ, spe2Δ, and spe3Δ). 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 spe2Δ 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.

Quantitative determination of aliphatic diamines and polyamines by an automated liquid chromatography procedure

Abstract The naturally occurring aliphatic polyamines and diamines can be separated with an improved automated procedure on a sulfonated-type ion-exchange column. Quantitative determinations can be carried out on 2–20 nmoles of each amine. In addition to the simple diamines, spermidine, and spermine, this procedure separates a number of naturally occurring derivatives, such as monoacetyl-1,4-diaminobutane, monoacetylspermidine, carbamyl-1,4-diaminobutane, 1,4-diamino-2-hydroxybutane, and glutathionylspermidine. The method can be used for a variety of biological materials, including urine, animal tissues, and bacterial extracts.

Spermidine but not spermine is essential for hypusine biosynthesis and growth in Saccharomyces cerevisiae: Spermine is converted to spermidine in vivo by the FMS1-amine oxidase

In our earlier work we showed that either spermidine or spermine could support the growth of spe2Δ or spe3Δ 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 spe3Δ fms1Δ 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.

Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase)

The Saccharomyces cerevisiae SPE3 gene, coding for spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3delta, which has no spermidine synthase activity and has an absolute requirement for spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of spermidine (10(-8) M) or a higher concentration of spermine (10(-6) M).

SPERMINE IS NOT ESSENTIAL FOR GROWTH OF SACCHAROMYCES CEREVISIAE : IDENTIFICATION OF THE SPE4 GENE (SPERMINE SYNTHASE) AND CHARACTERIZATION OF A SPE4 DELETION MUTANT

Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C. W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35-43].

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK)

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with Mrs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.

Polyamine deficiency leads to accumulation of reactive oxygen species in a spe2Δ mutant of Saccharomyces cerevisiae

We have previously shown that polyamine‐deficient Saccharomyces cerevisiae are very sensitive to incubation in oxygen. The current studies show that, even under more physiological conditions (i.e. growth in air), polyamine‐deficient cells accumulate reactive oxygen species (ROS). These cells develop an apoptotic phenotype and, after incubation in polyamine‐deficient medium, die. To show a specific effect of polyamines on ROS accumulation, uncomplicated by any effects on growth, spermine was added to spermidine‐deficient spe2Δ fms1Δ cells, since spermine does not affect the growth of this strain. In this strain, spermine addition caused a marked, but not complete, decrease in the accumulation of ROS and a moderate protection against cell death. In other experiments with polyamine‐deficient cells containing plasmids that overexpress superoxide dismutases (SOD1, SOD2), ROS decreased but with only a partial protection against cell death. Polyamine‐deficient cells incubated anaerobically show markedly less cell death. These data show that part of the function of polyamines is protection of the cells from accumulation of ROS. Copyright

Polyamines Are Not Required for Aerobic Growth of Escherichia coli: Preparation of a Strain with Deletions in All of the Genes for Polyamine Biosynthesis

A strain of Escherichia coli was constructed in which all of the genes involved in polyamine biosynthesis--speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), spe D (adenosylmethionine decarboxylase), speE (spermidine synthase), speF (inducible ornithine decarboxylase), cadA (lysine decarboxylase), and ldcC (lysine decarboxylase)--had been deleted. Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free medium, albeit at a slightly (ca. 40 to 50%) reduced growth rate. Even though this strain grew well in the absence of the amines in air, it was still sensitive to oxygen stress in the absence of added spermidine. In contrast to the ability to grow in air in the absence of polyamines, this strain, surprisingly, showed a requirement for polyamines for growth under strictly anaerobic conditions.

Absolute requirement of spermidine for growth and cell cycle progression of fission yeast (Schizosaccharomyces pombe)

Schizosaccharomyces pombe cells that cannot synthesize spermidine or spermine because of a deletion–insertion in the gene coding for S-adenosylmethionine decarboxylase (Δspe2) have an absolute requirement for spermidine for growth. Flow cytometry studies show that in the absence of spermidine an overall delay of the cell cycle progression occurs with some accumulation of cells in the G1 phase; as little as 10−6 M spermidine is sufficient to maintain normal cell cycle distribution and normal growth. Morphologically some of the spermidine-deprived cells become spherical at an early stage with little evidence of cell division. On further incubation in the spermidine-deprived medium, growth occurs in most of the cells, not by cell division but rather by cell elongation, with an abnormal distribution of the actin cytoskeleton, DNA (4′, 6-diamidino-2-phenylindole staining), and calcofluor-staining moieties. More prolonged incubation in the spermidine-deficient medium leads to profound morphological changes including nuclear degeneration.