Spermidine Synthase Research Articles

Alternative Spermidine Biosynthetic Route Is Critical for Growth of Campylobacter jejuni and Is the Dominant Polyamine Pathway in Human Gut Microbiota

The availability of fully sequenced bacterial genomes has revealed that many species known to synthesize the polyamine spermidine lack the spermidine biosynthetic enzymes S-adenosylmethionine decarboxylase and spermidine synthase. We found that such species possess orthologues of the sym-norspermidine biosynthetic enzymes carboxynorspermidine dehydrogenase and carboxynorspermidine decarboxylase. By deleting these genes in the food-borne pathogen Campylobacter jejuni, we found that the carboxynorspermidine decarboxylase orthologue is responsible for synthesizing spermidine and not sym-norspermidine in vivo. In polyamine auxotrophic gene deletion strains of C. jejuni, growth is highly compromised but can be restored by exogenous sym-homospermidine and to a lesser extent by sym-norspermidine. The alternative spermidine biosynthetic pathway is present in many bacterial phyla and is the dominant spermidine route in the human gut, stomach, and oral microbiomes, and it appears to have supplanted the S-adenosylmethionine decarboxylase/spermidine synthase pathway in the gut microbiota. Approximately half of the gut Firmicutes species appear to be polyamine auxotrophs, but all encode the potABCD spermidine/putrescine transporter. Orthologues encoding carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase are found clustered with an array of diverse putrescine biosynthetic genes in different bacterial genomes, consistent with a role in spermidine, rather than sym-norspermidine biosynthesis. Due to the pervasiveness of ε-proteobacteria in deep sea hydrothermal vents and to the ubiquity of the alternative spermidine biosynthetic pathway in that phylum, the carboxyspermidine route is also dominant in deep sea hydrothermal vents. The carboxyspermidine pathway for polyamine biosynthesis is found in diverse human pathogens, and this alternative spermidine biosynthetic route presents an attractive target for developing novel antimicrobial compounds.

Histatin 5 Uptake by Candida albicans Utilizes Polyamine Transporters Dur3 and Dur31 Proteins

Histatin 5 (Hst 5) is a salivary gland-secreted cationic peptide with potent fungicidal activity against Candida albicans. Hst 5 kills fungal cells following intracellular translocation, although its selective transport mechanism is unknown. C. albicans cells grown in the presence of polyamines were resistant to Hst 5 due to reduced intracellular uptake, suggesting that this cationic peptide may enter candidal cells through native yeast polyamine transporters. Based upon homology to known Saccharomyces cerevisiae polyamine permeases, we identified six C. albicans Dur polyamine transporter family members and propose a new nomenclature. Gene deletion mutants were constructed for C. albicans polyamine transporters Dur3, Dur31, Dur33, Dur34, and were tested for Hst 5 sensitivity and uptake of spermidine. We found spermidine uptake and Hst 5 mediated killing were decreased significantly in Δdur3, Δdur31, and Δdur3/Δdur31 strains; whereas a DUR3 overexpression strain increased Hst 5 sensitivity and higher spermidine uptake. Treatment of cells with a spermidine synthase inhibitor increased spermidine uptake and Hst 5 killing, whereas protonophores and cold treatment reduced spermidine uptake. Inhibition assays showed that Hst 5 is a competitive analog of spermidine for uptake into C. albicans cells, and that Hst 5 Ki values were increased by 80-fold in Δdur3/Δdur31 cells. Thus, Dur3p and Dur31p are preferential spermidine transporters used by Hst 5 for its entry into candidal cells. Understanding of polyamine transporter-mediated internalization of Hst 5 provides new insights into the uptake mechanism for C. albicans toxicity, and further suggests design for targeted fungal therapeutic agents.

Polyamines attenuate ethylene-mediated defense responses to abrogate resistance to Botrytis cinerea in tomato.

Transgenic tomato (Solanum lycopersicum) lines overexpressing yeast spermidine synthase (ySpdSyn), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild-type plants and were examined for responses to the fungal necrotrophs Botrytis cinerea and Alternaria solani, bacterial pathogen Pseudomonas syringae pv tomato DC3000, and larvae of the chewing insect tobacco hornworm (Manduca sexta). The Spd-accumulating transgenic tomato lines were more susceptible to B. cinerea than the wild-type plants; however, responses to A. solani, P. syringae, or M. sexta were similar to the wild-type plants. Exogenous application of ethylene precursors, S-adenosyl-Met and 1-aminocyclopropane-1-carboxylic acid, or PA biosynthesis inhibitors reversed the response of the transgenic plants to B. cinerea. The increased susceptibility of the ySpdSyn transgenic tomato to B. cinerea was associated with down-regulation of gene transcripts involved in ethylene biosynthesis and signaling. These data suggest that PA-mediated susceptibility to B. cinerea is linked to interference with the functions of ethylene in plant defense.

Chemopreventive mechanisms of methionine on inhibition of benzo(a)pyrene–DNA adducts formation in human hepatocellular carcinoma HepG2 cells

This study was designed to investigate the molecular mechanism underlying the chemopreventive effects of methionine on benzo[a]pyrene (B[a]P)–DNA adducts formation in HepG2 cells. Methionine significantly inhibited B[a]P–DNA adduct formation in HepG2 cells. Methionine significantly decreased the cellular uptake of [ 3H] B[a]P, but increased the cellular discharge of [ 3H] B[a]P from HepG2 cells into the media. B[a]P significantly lowered total cellular glutathione (GSH) level, but co-cultured with B[a]P and methionine, gradually attenuated intracellular GSH levels in a concentration-dependent manner, which was markedly higher at 20–500 μM methionine. The cellular proteins of treated cells were resolved by 2D-polyacrylamide gel electrophoresis. Proteomic profiles showed that phase II enzymes such as glutathione S-transferase (GST) omega-1, GSTM3, glyoxalase I (GLO1) and superoxide dismutase (SOD) were down-regulated by B[a]P treatment, whereas cathepsin B (CTSB), Rho GDP-dissociation inhibitor alpha (Rho-GDP-DIA), histamine N-methyltransferase (HNMT), spermidine synthase (SRM) and arginase-1 (ARG1) were up-regulated by B[a]P. B[a]P and methionine treatments, GST omega-1, GSTM3, GLO1 and SOD were significantly enhanced compared to B[a]P alone. Similarly, methionine was effective in diminishing the B[a]P-induced up-regulation of CTSB, Rho-GDP-DIA, HNMT, SRM and ARG1. Our data suggests that methionine might exert a chemoprotective effect on B[a]P–DNA adduct formation by attenuating intracellular GSH levels, blocking the uptake of B[a]P into cells, or by altering expression of proteins involved in DNA adduct formation.

Cell-autonomous circadian clock of hepatocytes drives rhythms in transcription and polyamine synthesis

The circadian clock generates daily rhythms in mammalian liver processes, such as glucose and lipid homeostasis, xenobiotic metabolism, and regeneration. The mechanisms governing these rhythms are not well understood, particularly the distinct contributions of the cell-autonomous clock and central pacemaker to rhythmic liver physiology. Through microarray expression profiling in Met murine hepatocytes (MMH)-D3, we identified over 1,000 transcripts that exhibit circadian oscillations, demonstrating that the cell-autonomous clock can drive many rhythms, and that MMH-D3 is a valid circadian model system. The genes represented by these circadian transcripts displayed both cophasic and antiphasic organization within a protein-protein interaction network, suggesting the existence of competition for binding sites or partners by genes of disparate transcriptional phases. Multiple pathways displayed enrichment in MMH-D3 circadian transcripts, including the polyamine synthesis module of the glutathione metabolic pathway. The polyamine synthesis module, which is highly associated with cell proliferation and whose products are required for initiation of liver regeneration, includes enzymes whose transcripts exhibit circadian oscillations, such as ornithine decarboxylase and spermidine synthase. Metabolic profiling revealed that the enzymatic product of spermidine synthase, spermidine, cycles as well. Thus, the cell-autonomous hepatocyte clock can drive a significant amount of transcriptional rhythms and orchestrate physiologically relevant modules such as polyamine synthesis.

Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine.

Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well-studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5'-deoxy-5'-methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S-adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose-dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X-ray crystallography at 2.0 Å resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K(d) of 1.1 ± 0.3 μM in the absence of putrescine and 3.2 ± 0.1 μM in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.

Characterization of transgenic mice with overexpression of spermidine synthase

A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to ninefold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5- to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N 1-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.

Spermidine Synthase Is Required for Virulence of Leishmania donovani

Genetic lesions in the polyamine biosynthetic pathway of Leishmania donovani, the causal agent of visceral leishmaniasis, are conditionally lethal mutations that render the insect vector form of the parasite auxotrophic for polyamines. Recently, we have demonstrated that a Δodc L. donovani null mutant lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was profoundly compromised in its ability to infect mice, indicating that ODC is essential for the infectious mammalian stage of the parasite and further validating the enzyme as a possible drug target. To assess whether other components of the polyamine biosynthetic pathway were also essential for parasite virulence, a cell line deficient in spermidine synthase (SPDSYN), the enzyme that converts putrescine to spermidine, was created by double-targeted gene replacement within a virulent L. donovani background. This Δspdsyn strain was auxotrophic for polyamines, required spermidine for growth in its insect vector form, and was adversely impacted in its ability to infect mice. These findings establish that SPDSYN, like ODC, is essential for maintaining a robust infection in mammals and indicate that pharmacologic inhibition of SPDSYN, and perhaps all components of the polyamine biosynthetic pathway, is a valid therapeutic strategy for the treatment of visceral and, potentially, other forms of leishmaniasis.

Effects of salt and osmotic stresses on free polyamine content and expression of polyamine biosynthetic genes in Vitis vinifera

Grape (Vitis vinifera L.) seedlings grown in vitro were treated with either 200 mM NaCl or 350 mM mannitol for 7 d. Both salinity and osmotic stress caused significant increase in electrolyte leakage. From the three commonly occurring free polyamines (PA), only conspicuous accumulation of putrescine was found in the NaCl-treated seedlings. Four PA biosynthetic genes encoding arginine decarboxylase (pVvADC), S-adenosylmethionine decarboxylase (pVvSAMDC), spermidine synthase (pVvSPDS) and spermine synthase (pVvSPMS) were successfully isolated. While induction of pVvADC was observed from the 1st day of salt treatment, pVvSAMDC and pVvSPMS were induced only at late stage of stress. As for expression levels of genes in the mannitol-treated seedling, either temporary (pVvADC at day 1) or late (pVvSPMS at days 5 and 7) induction was observed.

Crystal Structures and Enzymatic Properties of a Triamine/Agmatine Aminopropyltransferase from Thermus thermophilus

To maintain functional conformations of DNA and RNA in high-temperature environments, an extremely thermophilic bacterium, Thermus thermophilus, employs a unique polyamine biosynthetic pathway and produces more than 16 types of polyamines. In the thermophile genome, only one spermidine synthase homolog (SpeE) was found and it was shown to be a key enzyme in the pathway. The catalytic assay of the purified enzyme revealed that it utilizes triamines (norspermidine and spermidine) and agmatine as acceptors in its aminopropyl transfer reaction; therefore, the enzyme was denoted as a triamine/agmatine aminopropyltransferase (TAAPT). We determined the crystal structures of the enzyme complexed with and without the aminopropyl group donor S-adenosylmethionine. Despite sequence and structural similarity with spermidine synthases from other organisms, a novel C-terminal β-sheet and differences in the catalytic site were observed. The C-terminal module interacts with the gatekeeping loop and fixes the open conformation of the loop to recognize larger polyamine substrates such as agmatine and spermidine. Additional computational docking studies suggest that the structural differences of the catalytic site also contribute to recognition of the aminopropyl/aminobutyl or guanidium moiety of the substrates of TAAPT. These results explain in part the extraordinarily diverse polyamine spectrum found in T. thermophilus.

Ectopic expression of MdSPDS1 in sweet orange (Citrus sinensis Osbeck) reduces canker susceptibility: involvement of H₂O₂ production and transcriptional alteration.

BackgroundEnormous work has shown that polyamines are involved in a variety of physiological processes, but information is scarce on the potential of modifying disease response through genetic transformation of a polyamine biosynthetic gene.ResultsIn the present work, an apple spermidine synthase gene (MdSPDS1) was introduced into sweet orange (Citrus sinensis Osbeck 'Anliucheng') via Agrobacterium-mediated transformation of embryogenic calluses. Two transgenic lines (TG4 and TG9) varied in the transgene expression and cellular endogenous polyamine contents. Pinprick inoculation demonstrated that the transgenic lines were less susceptible to Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker, than the wild type plants (WT). In addition, our data showed that upon Xac attack TG9 had significantly higher free spermine (Spm) and polyamine oxidase (PAO) activity when compared with the WT, concurrent with an apparent hypersensitive response and the accumulation of more H2O2. Pretreatment of TG9 leaves with guazatine acetate, an inhibitor of PAO, repressed PAO activity and reduced H2O2 accumulation, leading to more conspicuous disease symptoms than the controls when both were challenged with Xac. Moreover, mRNA levels of most of the defense-related genes involved in synthesis of pathogenesis-related protein and jasmonic acid were upregulated in TG9 than in the WT regardless of Xac infection.ConclusionOur results demonstrated that overexpression of the MdSPDS1 gene prominently lowered the sensitivity of the transgenic plants to canker. This may be, at least partially, correlated with the generation of more H2O2 due to increased production of polyamines and enhanced PAO-mediated catabolism, triggering hypersensitive response or activation of defense-related genes.

Antisense inhibition of a spermidine synthase gene highlights the role of polyamines for stress alleviation in pear shoots subjected to salinity and cadmium

Three transgenic European pear ( Pyrus communis L.) lines with reduced spermidine synthase ( SPDS) expression and spermidine (Spd) titers were developed using a construct containing an apple SPDS gene ( MdSPDS1) in antisense orientation. After exposure to either salt or cadmium stress, growth inhibition was more severe in the antisense lines than in the wild-type (WT). The antioxidant system, as shown by glutathione (GSH) content, activity of glutathione reductase (GR) and superoxide dismutase (SOD), and proline accumulation, was not effectively induced under stress in the antisense lines as compared with the WT. The reduction in antioxidant system function in the antisense lines was accompanied by a greater accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation. Growth inhibition, Spd level, and parameters indicative of the antioxidant system were significantly ameliorated by exogenous Spd application. Under either salt or cadmium stress, GSH content, GR and SOD activity, and proline accumulation were positively correlated with Spd, putrescine (Put), and total polyamine titers. Conversely, MDA level showed a significantly negative correlation with these polyamines under both stress conditions. Thus, the responses to stress treatments were first identified in the SPDS antisense European pears, and the results provide further evidence for the important role of polyamines in both salt and cadmium stress tolerance, in which the polyamines act, at least in part, by influencing the antioxidant system.

Polyamine metabolic canalization in response to drought stress in Arabidopsis and the resurrection plant Craterostigma plantagineum

In this work, we have studied the transcriptional profiles of polyamine biosynthetic genes and analyzed polyamine metabolic fluxes during a gradual drought acclimation response in Arabidopsis thaliana and the resurrection plant Craterostigma plantagineum. The analysis of free putrescine, spermidine and spermine titers in Arabidopsis arginine decarboxylase (adc1-3, adc2-3), spermidine synthase (spds1-2, spds2-3) and spermine synthase (spms-2) mutants during drought stress, combined with the quantitative expression of the entire polyamine biosynthetic pathway in the wild-type, has revealed a strong metabolic canalization of putrescine to spermine induced by drought. Such canalization requires spermidine synthase 1 (SPDS1) and spermine synthase (SPMS) activities and, intriguingly, does not lead to spermine accumulation but to a progressive reduction in spermidine and spermine pools in the wild-type. Our results suggest the participation of the polyamine back-conversion pathway during the drought stress response rather than the terminal catabolism of spermine. The putrescine to spermine canalization coupled to the spermine to putrescine back-conversion confers an effective polyamine recycling-loop during drought acclimation. Putrescine to spermine canalization has also been revealed in the desiccation tolerant plant C. plantagineum, which conversely to Arabidopsis, accumulates high spermine levels which associate with drought tolerance. Our results provide a new insight to the polyamine homeostasis mechanisms during drought stress acclimation in Arabidopsis and resurrection plants.

Identification of differentially-expressed genes potentially related to stress tolerance in a transgenic line of European pear over-expressing an apple spermidine synthase gene (MdSPDS1)

SummaryPolyamines confer abiotic stress tolerance through mechanisms that are not yet fully understood. Previous studies on a transgenic line of European pear (Pyrus communis L., line #32), over-expressing an apple gene for spermidine synthase (MdSPDS1), provided greater insight into the mechanisms of stress tolerance, leading to agronomic benefits, as well as being an elite line to be used to further elucidate the molecular mechanisms underlying stress tolerance that are exerted by spermidine (Spd). In this study, a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) method, involving an annealing control primer (ACP)-based PCR (GeneFishing) system, was employed to identify genes that may be implicated in abiotic stress tolerance. A total of 42 transcripts that were differentially expressed between line #32 and wild-type (WT) pear were identified. After cloning, 24 of these were found to exhibit significant sequence similarities with known genes from other species, and the remaining 18 were annotated as “unknown”. To verify the reliability of the ACP-based differential display method, ten clones (five up-regulated and five down-regulated in line #32 compared to WT) were subjected to real-time RT-PCR analysis. These clones were further analysed to determine the relative abundance of the target sequences under conditions of salinity or cadmium stress. The results indicated that GeneFishing is a useful method to identify differentially-expressed genes (DEGs) in an MdSPDS1-overexpressing transgenic line of pear. The possible involvement of these DEGs in salinity and cadmium stress tolerance is also discussed based on an analysis of their levels of expression.

Deepening into the proteome of maize cells habituated to the cellulose biosynthesis inhibitor dichlobenil

Cellulose biosynthesis inhibitors, such as dichlobenil (DCB), have become a valuable tool for the analysis of structural and compositional plasticity of plant cell walls. By stepwise increasing the concentration of DCB in the culture medium, we obtained maize cells able to cope with DCB through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of feruloylated arabinoxylans. Recently we demonstrated that the expression of several Cellulose Synthase and phenylpropanoid-related genes is altered in DCB-habituated cells. In addition, by using a proteomic approach we identified several proteins induced or repressed in DCB-habituated cells. After a more in-depth analysis, some new proteins induced (two inhibitors TAXI-IV, an α-1,4-glucan-protein synthase, and a pectinesterase inhibitor) or repressed (a chaperonin 60, a fructokinase-1 and a spermidine synthase 1) were identified, and their possible role in the context of DCB-habituation is discussed.

Overexpression of apple spermidine synthase 1 (MdSPDS1) leads to significant salt tolerance in tomato plants

The aim of this research is to study the effects of salt stress during different stages of development in transgenic tomato plants overexpressing the apple spermidine synthase gene (MdSPDS1) compared to wild type (WT) plants. Under salt treatment (100 and 150 mM NaCl), tomato plants clearly displayed several stress symptoms such as impaired seedling growth, decreased chlorophyll content, reduction in fruit yield and increased electrolyte leakage (EL) in leaves. These changes were more prominent in WT plants compared to MdSPDS1 transgenic plants which accumulate significantly more polyamines, namely spermine and spermidine. The response of ascorbate peroxidase (APX) isoenzymes in tomato leaves under saline conditions was also investigated. The transcript levels of SlApx genes were significantly up regulated under 100 mM NaCl either in wild type or in transgenic plants. Under 150 mM NaCl, only transgenic plants were capable to maintain high expression of SlApx genes, whereas in WT plants the expression declined after one month treatment. As a consequence, APX activity was significantly higher in MdSPDS1 transgenic plants compared to WT under saline conditions. These results suggest that elevated APX activity might contribute largely to the protection against oxidative stress generated by NaCl treatments and thus confer salinity tolerance in tomato overexpressing MdSPDS1.

Spermidine Is a Morphogenetic Determinant for Cell Fate Specification in the Male Gametophyte of the Water FernMarsilea vestita

Here, we show that the polyamine spermidine plays a key role as a morphogenetic determinant during spermatid development in the water fern Marsilea vestita. Spermidine levels rise first in sterile jacket cells and then increase dramatically in spermatogenous cells as the spermatids mature. RNA interference and drug treatments were employed to deplete spermidine in the gametophyte at different stages of gametogenesis. Development in spermidine-depleted gametophytes was arrested before the completion of the last round of cell divisions. In spermidine-depleted spermatogenous cells, chromatin failed to condense properly, basal body positioning was altered, and the microtubule ribbon was in disarray. When cyclohexylamine, a spermidine synthase (SPDS) inhibitor, was added at the start of spermatid differentiation, the spermatid nuclei remained round, centrin failed to localize into basal bodies, thus blocking basal body formation, and the microtubule ribbon was completely abolished. In untreated gametophytes, spermidine made in the jacket cells moves into the spermatids, where it is involved in the unmasking of stored SPDS mRNAs, leading to substantial spermidine synthesis in the spermatids. We found that treating spores directly with spermidine or other polyamines was sufficient to unmask a variety of stored mRNAs in gametophytes and arrest development. Differences in patterns of transcript distribution after these treatments suggest that specific transcripts reside in different locations in the dry spore; these differences may be linked to the timing of unmasking and translation for that mRNA during development.

Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells

Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra×maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the regulation of this pathway.

Polyamine biosynthesis and transport mechanisms are crucial for fitness and pathogenesis of Streptococcus pneumoniae

Polyamines such as cadaverine, putrescine and spermidine are polycationic molecules that have pleiotropic effects on cells via their interaction with nucleic acids. Streptococcus pneumoniae (the pneumococcus) is a Gram-positive pathogen capable of causing pneumonia, septicaemia, otitis media and meningitis. Pneumococci have a polyamine transport operon (potABCD) responsible for the binding and transport of putrescine and spermidine, and can synthesize cadaverine and spermidine using their lysine decarboxylase (cad) and spermidine synthase (speE) enzymes. Previous studies from our laboratory have shown that an increase in PotD expression is seen following exposure to various stresses, while during infection, potD inactivation significantly attenuates pneumococcal virulence, and anti-PotD immune responses are protective in mice. In spite of their relative importance, not much is known about the global contribution of polyamine biosynthesis and transport pathways to pneumococcal disease. Mutants deficient in polyamine biosynthesis (ΔspeE or Δcad) or transport genes (ΔpotABCD) were constructed and were found to be attenuated in murine models of pneumococcal colonization and pneumonia, either alone or in competition with the wild-type strain. The ΔspeE mutant was also attenuated during invasive disease, while the potABCD and cad genes seemed to be dispensable. HPLC analyses showed reduced intracellular polyamine levels in all mutant strains compared with wild-type bacteria. High-throughput proteomic analyses indicated reduced expression of growth, replication and virulence factors in mutant strains. Thus, polyamine biosynthesis and transport mechanisms are intricately linked to the fitness, survival and pathogenesis of the pneumococcus in host microenvironments, and may represent important targets for prophylactic and therapeutic interventions.