Manas K. Chattopadhyay

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.

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.

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.

Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well‐established in vivo. In this work we have performed microarray experiments with a spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10−5 M) or spermine (10−5 M) compared to a control culture containing 10−8 M spermidine. 247 genes were upregulated > two‐fold and 11 genes were upregulated >10‐fold after spermidine addition. Functional categorization of the genes showed induction of transport‐related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two‐fold, and six genes were downregulated > eight‐fold after spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. Copyright

Polyamines Are Critical for the Induction of the Glutamate Decarboxylase-dependent Acid Resistance System in Escherichia coli

Background: Polyamines are present in all organisms. Results: Polyamines induce various components of the glutamate-dependent acid resistance pathway (GDAR) in Escherichia coli and are important for protection against acid stress. Conclusion: A unique function of polyamines is the induction of the GDAR system. Significance: Polyamines are important for the survival of Escherichia coli when passing through the acid environment of the stomach. As part of our studies on the biological functions of polyamines, we have used a mutant of Escherichia coli that lacks all the genes for polyamine biosynthesis for a global transcriptional analysis on the effect of added polyamines. The most striking early response to the polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR) that is important for the survival of the bacteria when passing through the acid environment of the stomach. Not only were the two genes for glutamate decarboxylases (gadA and gadB) and the gene for glutamate-γ-aminobutyrate antiporter (gadC) induced by the polyamine addition, but the various genes involved in the regulation of this system were also induced. We confirmed the importance of polyamines for the induction of the GDAR system by direct measurement of glutamate decarboxylase activity and acid survival. The effect of deletions of the regulatory genes on the GDAR system and the effects of overproduction of two of these genes were also studied. Strikingly, overproduction of the alternative σ factor rpoS and of the regulatory gene gadE resulted in very high levels of glutamate decarboxylase and almost complete protection against acid stress even in the absence of any polyamines. Thus, these data show that a major function of polyamines in E. coli is protection against acid stress by increasing the synthesis of glutamate decarboxylase, presumably by increasing the levels of the rpoS and gadE regulators.

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: Purification and characterization of the inhibitory complex

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K(A)) between yeast AZ and yeast ODC is 6×10(7) (M(-1)). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.