The genes mnmE and mnmG are responsible for the modification of uridine 34, 'the wobble position' of many aminoacyl-tRNAs. Deletion of these genes affects the strength of the codon-anticodon interactions of the aminoacyl-tRNAs with the mRNAs and the ribosomes. However, deletion of these genes does not usually have a significant effect on the growth rate of the standard Escherichia coli strains. In contrast, we have found that if the host E. coli strain is deficient in the synthesis of polyamines, deletion of the mnmE or mnmG gene results in complete inhibition of growth unless the medium contains polyamines. The finding of an absolute requirement for polyamines in our current work will be significant in studies on polyamine function, in studies on the function of the mnmE/G genes, and in studies on the role of aminoacyl-tRNAs in protein biosynthesis.

To study the physiological roles of polyamines, we carried out a global microarray analysis on the effect of adding polyamines to an Escherichia coli mutant that lacks polyamines because of deletions in the genes in the polyamine biosynthetic pathway. Previously, we have reported that the earliest response to polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR). We also presented preliminary evidence for the involvement of rpoS and gadE regulators. In the current study, further confirmation of the regulatory roles of rpoS and gadE is shown by a comparison of genome-wide expression profiling data from a series of microarrays comparing the genes induced by polyamine addition to polyamine-free rpoS(+)/gadE(+) cells with genes induced by polyamine addition to polyamine-free Delta rpoS/gadE(+) and rpoS6+/Delta gadE cells. The results indicate that most of the genes in the E. coli GDAR system that are induced by polyamines require rpoS and gadE. Our data also show that gadE is the main regulator of GDAR and other acid fitness island genes. Both polyamines and rpoS are necessary for the expression of gadE gene from the three promoters of gadE (P1, P2, and P3). The most important effect of polyamine addition is the very rapid increase in the level of RpoS sigma factor. Our current hypothesis is that polyamines increase the level of RpoS protein and that this increased RpoS level is responsible for the stimulation of gadE expression, which in turn induces the GDAR system in E. coli.

Glutathionylspermidine synthetase/amidase (Gss) and the encoding gene (gss) have only been studied in Escherichia coli and several members of the Kinetoplastida phyla. In the present article, we have studied the phylogenetic distribution of Gss and have found that Gss sequences are largely limited to certain bacteria and Kinetoplastids and are absent in a variety of invertebrate and vertebrate species, Archea, plants, and some Eubacteria. It is striking that almost all of the 75 Enterobacteria species that have been sequenced contain sequences with very high degree of homology to the E. coli Gss protein. To find out the physiological significance of glutathionylspermidine in E. coli, we have performed global transcriptome analyses. The microarray studies comparing gss(+) and Δgss strains of E. coli show that a large number of genes are either up-regulated (76 genes more than threefold) or down-regulated (35 genes more than threefold) by the loss of the gss gene. Most significant categories of up-regulated genes include sulfur utilization, glutamine and succinate metabolism, polyamine and arginine metabolism, and purine and pyrimidine metabolism.

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). In the present study we have purified yeast antizyme and ornithine decarboxylase and we have studied the biochemical and biophysical characteristics of the proteins, and the resulting ODC:AZ complex. The surface plasmon resonance analyses show association constant (KA) between yeast AZ to yeast ODC is 6x 107 (M−1), which is ~1/1000th as determined in case of mammalian ODC‐ AZ interaction. The circular dichroism (CD) spectra of the two individual proteins in different ratios and of the ODC:AZ complex show no significant change in the secondary or tertiary structure of either ODC or AZ after binding to each other. The CD spectra also show that the secondary structure of yeast antizyme is different from the published NMR structure of rat antizyme. Using purified His‐tagged AZ as a binding partner, we have purified the ODC: AZ heterodimer and show that it has no ODC activity. The native molecular weight of the complex is 90 kDa, which suggests a one is to one stoichiometric binding of AZ and ODC in vitro.

ABSTRACT 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.

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 Δ, spe 2Δ, and spe3 Δ ) . 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 Δ 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.

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 spe2 Δ fms1 Δ 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 © 2006 John Wiley & Sons, Ltd.