Metabolic discrimination of sucrose starvation from<Emphasis Type="Italic">Arabidopsis</Emphasis> cell suspension by<Superscript>1</Superscript>H NMR based metabolomics

Whole cell extracts ofArabidopsis cell cultures maintained on various sucrose concentrations (0,3, and 6%) were analyzed by¹H NMR spectroscopy to determine the comprehensive metabolic change in these cultures during sucrose starvation. The amount of sucrose, glucose, and fructose in the cells decreased to almost nothing after 12 h of culture in medium without sucrose. In contrast, the total free amino acid content of the cells increased as the culture proceeded. Among the free amino acids, phenylalanine and malic acid increased the most, followed by asparagine and alanine, whereas glutamic acid did not change significantly. These results are in agreement with previous studies using HPLC.¹H NMR spectroscopy enabled measurement of changes in the sugar and free amino acid content of whole cell extracts without fractionation and complicated sample preparation. These results indicate that comprehensive metabolic changes in the cells can be determined by a simple, rapid method using whole cell extracts and¹H NMR spectroscopy.     

Requirement of Polyamines for Bacterial Division

Synchronous cell division in an arginine auxotroph and a histidine auxotroph of Escherichia coli was obtained after starving for the required amino acid for 1 hr. However, cell division was not synchronized after starvation for 1 hr in another arginine auxotroph. This difference is proposed to depend on differences in the concentrations of polyamines in the cells. During amino acid starvation the ratio of putrescine concentration to spermidine concentration decreased in all strains, but it recovered afterward more rapidly in the third strain than in the other two. The cells divided when the ratio returned to normal in the Arg(-) mutants. Added putrescine permitted some of the cells of the first two mutants to divide sooner after amino acid starvation and thus eliminated synchrony. Spermidine added alone had no effect, but, when it was added together with putrescine, it restored synchronous division. Synchrony was established in the third mutant by adding spermidine after arginine starvation. Thus, both the variations in polyamine content and the effects of added polyamines suggest that the polyamines are essential in permitting cell division. We suggest that the molar ratio of putrescine to spermidine can be a critical factor for cell division. This effect of polyamines seems to be specific for cell division. Amino acid starvation does not induce delays in subsequent mass increase or deoxyribonucleic acid synthesis. Possible mechanisms of polyamine action are discussed.     

Role of relA mutation in the survival of amino acid-starved Escherichia coli

Amino acid-starved cells of Escherichia coli relA ⁺, which contain a large number of glycogen particles, are able to survive in phosphate buffer for a longer time period than their relaxed counterparts. With regard to NH ₄ ⁺ starvation differences in the survival of both strains were not found. NH ₄ ⁺ starved cells of E. coli relA are able to synthesize glycogen but amino acid-starved cells of the relA strain are not. We suggest that the synthesis of glycogen triggered by guanosine tetraphosphate during amino acid starvation is responsible for the prolonged viability of the E. coli relA ⁺ strain.Abbreviations ppGpp guanosine tetraphosphate     

Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction

Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.     

Abnormal growth of polyamine-deficient Escherichia coli mutant is partially caused by oxidative stress-induced damage

Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli. In this study, we constructed a new polyamine-deficient E. coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions. We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth. Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including z.rad;O(2)(-), and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner. As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type. Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant. Finally, we confirmed that chloromethyl-2('),7(')-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells. These results showed that abnormal growth of the polyamine-deficient E. coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth.     

What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome?

The Escherichia coli K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The mazEF and relBE systems have been extensively characterized and were proposed to be general stress response modules. On one hand, mazEF was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand, relBE and mazEF were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized E. coli TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.     

Effect of N 1-guanyl-1,7-diaminoheptane,an inhibitor of deoxyhypusine synthase,on endothelial cell growth,differentiation and apoptosis

An unusual amino acid, hypusine [N -(4-amino-2-hydroxybutyl)lysine], is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A (eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although eIF5A and its hypusine modification are essential for eukaryotic cell viability, the true physiological function of eIF5A is yet unknown. We have examined the effects of N ¹-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor of deoxyhypusine synthase, on endothelial cell proliferation, differentiation and apoptosis. Upon treatment of human umbilical vein endothelial cells (HUVEC) with GC7, dose-dependent inhibition of hypusine formation and cellular proliferation was observed. GC7 at 10 M caused almost complete inhibition of cellular hypusine synthesis and led to cytostasis of HUVEC. Pretreatment of HUVEC with GC7 up to 50 M for 4 days had little effect on the attachment and differentiation of these cells on Matri-gel and did not cause induction of apoptosis. Instead, the GC7 pretreatment (96 h at 5–50 M) elicited protective effects against apoptotic death of HUVEC induced by serum starvation. These results suggest that eIF-5A may be involved in expression of proteins essential for apoptosis of endothelial cells as well as those for cellular proliferation.     

Genome-wide mRNA profiling in glucose starved <Emphasis Type="Italic">Bacillus subtilis</Emphasis> cells

In this study global changes in gene expression were monitored in Bacillus subtilis cells entering stationary growth phase owing to starvation for glucose. Gene expression was analysed in growing and starving cells at different time points by full-genome mRNA profiling using DNA macroarrays. During the transition to stationary phase we observed extensive reprogramming of gene expression, with ~1000 genes being strongly repressed and ~900 strongly up-regulated in a time-dependent manner. The genes involved in the response to glucose starvation can be assigned to two main classes: (i) general stress/starvation genes which respond to various stress or starvation stimuli, and (ii) genes that respond specifically to starvation for glucose. The first class includes members of the ^B-dependent general stress regulon, as well as 90 vegetative genes, which are strongly down regulated in the course of the stringent response. Among the genes in the second class, we observed a decrease in the expression of genes encoding proteins required for glucose uptake, glycolysis and the tricarboxylic acid cycle. Conversely, many carbohydrate utilisation systems that depend on phosphotransferase systems (PTS) or ABC transporters were activated. The expression of genes required for utilisation or generation of acetate indicates that acetate constitutes an important energy source for B. subtilis during periods of glucose starvation. Finally, genome wide mRNA profiling data can be used to predict new metabolic pathways in B. subtilis. Thus, our data suggest that glucose-starved cells are able to degrade branched-chain fatty acids to pyruvate and succinate via propionyl-CoA using the methylcitrate pathway. This pathway appears to link lipid degradation to gluconeogenesis in glucose-starved cells.This revised version was published online in May 2005 with corrections to the list of authors     

Stabilization of glucose-starved Escherichia coli K12 and Salmonella typhimurium LT2 by peptidase-deficient mutants

Escherichia coli K12 and Salmonella typhimurium LT2 cells were stabilized during carbon starvation in the presence of peptidase-deficient mutant strains. The rate of loss of viability of the wild-type S. typhimurium strain was decreased an average of 2-fold, and the rate for the wild-type E. coli strain was decreased about 2.3-fold, when either was starved in the presence of the multiply peptidase-deficient S. typhimurium strain TN852; other peptidase-deficient strains exhibited similar stabilizing effects. Starving wild-type S. typhimurium LT2 cells utilized peptides excreted by the starving peptidase-deficient cells for protein synthesis, and, to a lesser extent, as respiratory substrates. Provision of free amino acids in steady-state levels to starving E. coli K12 cells in a cell recycle apparatus had a stabilizing effect similar to that of mixing with peptidase-deficient cells.     

Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation

The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the same levels as found during growth.Nonstandard Abbreviations cGMP 3,5 guanosine monophosphate - ppGpp guanosine tetraphosphate - MS mineral salts - HC casein hydrolysate - TEA triethanolamine buffer - Pi inorganic phosphate     

Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures

Association of the availability of nutrients with the phenomenon of programmed cell death-apoptosis-was investigated using hybridoma cells cultured in protein-free medium under conditions of starvation, i.e., in RPMl-1640 medium diluted to 50% with saline. Amino acid mixtures, such as MEM essential amino acids or MEM nonessential amino acids were found to prevent starvation death significantly when added to the diluted medium in 1 to 2 mM concentrations, the MEM vitamin mixture was ineffective, and glutamine displayed a moderate growth-supporting effect. The specific monoclonal antibody production rate in cultures supplemented with amino acid mixtures was strikingly low, whereas supplementation with glutamine alone or simultaneously with other amino acids resulted in a specific antibody production rate comparable with the rate observed in undiluted medium. (c) 1995 John Wiley & Sons, Inc.     

The Proteomic Response of Bacillus pumilus Cells to Glucose Starvation

Since starvation for carbon sources is a common condition for bacteria in nature and it can also occur in industrial fermentation processes due to mixing zones, knowledge about the response of cells to carbon starvation is beneficial. The preferred carbon source for bacilli is glucose. The response of Bacillus pumilus cells to glucose starvation using metabolic labeling and quantitative proteomics was analyzed. Glucose starvation led to an extensive reprogramming of the protein expression pattern in B. pumilus. The amounts of proteins of the central carbon metabolic pathways (glycolysis and TCC) remained stable in starving cells. Proteins for gluconeogenesis were found in higher amounts during starvation. Furthermore, many proteins involved in acquisition and usage of alternative carbon sources were present in elevated amounts in starving cells. Enzymes for fatty acid degradation and proteases and peptidases were also found in higher abundance when cells entered stationary phase. Among the proteins found in lower amounts were many enzymes involved in amino acid and nucleotide synthesis and several NRPS and PKS proteins.     

Survival of Streptococcus pyogenes under stress and starvation

The ability of Streptococcus pyogenes to enter a quiescent state, similar to the stationary phase of lab cultures, is believed to be an important factor in its ability to persist within the host and to subsequently cause disease. Using a model broth system, we determined that after entering the stationary phase, there was a 99.99% reduction in cell viability over a 4-day period, following which the cells appeared to enter a resistant starvation state where cell numbers remained constant over the subsequent 3-4 weeks. This starvation response was induced by carbon or phosphorous limitation, but not by nitrogen limitation in the form of amino acids where cells became non-culturable after 4 days. Amino acid utilization in the absence of a carbon source may be an essential factor for the long-term survival of this bacterium in the stationary phase. Early stationary phase cells showed a greater resistance to oxidative and pH stress compared to 24-h-starved cultures. There was evidence for the formation of a viable but non-culturable state as indicated by a comparison of the numbers of cells with a functional membrane potential (rhodamine 123) against culturable cells on either Todd Hewitt broth agar or sheep blood agar. Long-term survival of S. pyogenes was dependent on both cell wall and protein synthesis, suggesting that starving cultures are a dynamic cell population.     

Survival Kinetics of Starving Bacteria Is Biphasic and Density-Dependent

In the lifecycle of microorganisms, prolonged starvation is prevalent and sustaining life during starvation periods is a vital task. In the literature, it is commonly assumed that survival kinetics of starving microbes follows exponential decay. This assumption, however, has not been rigorously tested. Currently, it is not clear under what circumstances this assumption is true. Also, it is not known when such survival kinetics deviates from exponential decay and if it deviates, what underlying mechanisms for the deviation are. Here, to address these issues, we quantitatively characterized dynamics of survival and death of starving E. coli cells. The results show that the assumption – starving cells die exponentially – is true only at high cell density. At low density, starving cells persevere for extended periods of time, before dying rapidly exponentially. Detailed analyses show intriguing quantitative characteristics of the density-dependent and biphasic survival kinetics, including that the period of the perseverance is inversely proportional to cell density. These characteristics further lead us to identification of key underlying processes relevant for the perseverance of starving cells. Then, using mathematical modeling, we show how these processes contribute to the density-dependent and biphasic survival kinetics observed. Importantly, our model reveals a thrifty strategy employed by bacteria, by which upon sensing impending depletion of a substrate, the limiting substrate is conserved and utilized later during starvation to delay cell death. These findings advance quantitative understanding of survival of microbes in oligotrophic environments and facilitate quantitative analysis and prediction of microbial dynamics in nature. Furthermore, they prompt revision of previous models used to analyze and predict population dynamics of microbes.     

Physiological responses of Lactococcus lactis ML3 to alternating conditions of growth and starvation

Lactococcus lactis species can survive periods of carbohydrate starvation for relatively long periods of time. In the first hours of starvation, however, the maximal glycolytic and arginine deiminase (ADI) pathway activities decline rapidly. The rate of decrease of the pathway activities diminishes as soon as the cells become depleted of energy-rich intermediates. Loss of glycolytic activity is associated with loss of glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase and pyruvate kinase activities. Upon addition of sugar to starved cultures these enzymatic, and thus the glycolytic, activities can be restored to 100% values. The recovery of enzymatic activities is inhibited by chloramphenicol, indicating that protein synthesis is involved. In contrast, restoration of ADI pathway activity does not require de novo synthesis of proteins. General protein degradation and synthesis have been studied in growing and starving cells using [³⁵S]methionine-labeling of proteins and two-dimensional gel analysis. The breakdown of bulk proteins in exponentially growing cells shows first-order rate kinetics (t1/2 of approximately 5 h). Following an initial breakdown of proteins with a t1/2 of 5 h during the first hour(s) of starvation, bulk proteins are degraded very slowly in starving energy-depleted cells. The breakdown of proteins during starvation appears to be (largely) nonspecific. The rate of synthesis of proteins decreases rapidly in the first hour(s) of starvation. From the onset of starvation on at least 45 proteins are no longer synthesized. During starvation relative induction of fourteen to fifteen proteins can be observed.Abbreviations ADI Arginine deiminase - ATP adenosine triphosphate - PEP phosphoenolpyruvate - membrane potential - pH pH gradient - PTS sugar phosphotransferase system - CDM chemically defined medium - TCA trichloro-acetic acid     

Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis

The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al. (1994), J. Cell Sci. 107, 2691-2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely resembles apoptosis of most mammalian cells, involves loss of mitochondrial transmembrane potential, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning or transmission electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a "conditioned" cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed.     

Mitochondrial function in Parkinson's disease cybrids containing an nt2 neuron-like nuclear background

Mitochondria likely play a role in Parkinson's disease (PD) neurodegeneration. We modelled PD by creating cytoplasmic hybrid (cybrid) cell lines in which endogenous mitochondrial DNA (mtDNA) from PD or control subject platelets was expressed within human teratocarcinoma (NT2) cells previously depleted of endogenous mtDNA. Complex I activity was reduced in both PD cybrid lines and in the platelet mitochondria used to generate them. Under basal conditions PD cybrids had less ATP, more LDH release, depolarized mitochondria, less mitochondrial cytochrome c, and higher caspase 3 activity. Equivalent MPP+ exposures are more likely to trigger programmed cell death in PD cybrid cells than in control cybrid cells. Our data support a relatively upstream role for mitochondrial dysfunction in idiopathic PD.