Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Synthesis of the Arabidopsis bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase enzyme of lysine catabolism is concertedly regulated by metabolic and stress-associated signals

In plants, excess cellular lysine (Lys) is catabolized into glutamic acid and acetyl-coenzyme A; yet, it is still not clear whether this pathway has other functions in addition to balancing Lys levels. To address this issue, we examined the effects of stress-related hormones, abscisic acid (ABA), and jasmonate, as well as various metabolic signals on the production of the mRNA and polypeptide of the bifunctional Lys-ketoglutarate reductase (LKR)/saccharopine dehydrogenase (SDH) enzyme, which contains the first two linked enzymes of Lys catabolism. The level of LKR/SDH was strongly enhanced by ABA, jasmonate, and sugar starvation, whereas excess sugars and nitrogen starvation reduced its level; thus this pathway appears to fulfill multiple functions in stress-related and carbon/nitrogen metabolism. Treatments with combination of hormones and/or metabolites, as well as use of ABA mutants in conjunction with the tester sugars mannose and 3-O-methyl-glucose further supported the idea that the hormonal and metabolic signals apparently operate through different signal transduction cascades. The stimulation of LKR/SDH protein expression by ABA is regulated by a signal transduction cascade that contains the ABI1-1 and ABI2-1 protein phosphatases. By contrast, the stimulation of LKR/SDH protein expression by sugar starvation is regulated by the hexokinase-signaling cascade in a similar manner to the repression of many photosynthetic genes by sugars. These findings suggest a metabolic and mechanistic link between Lys catabolism and photosynthesis-related metabolism in the regulation of carbon/nitrogen partitioning.     

The effect of nucleosides on the induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli

The induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli starving for exogenous carbon and nitrogen sources was stimulated markedly by the addition of any of four nucleosides tested: adenosine, guanosine, cytidine, and uridine. Adenosine was used as a representative of this group of compounds in most experiments. The decrease of ability of the cells to synthesize β-galactosidase, resulting from a prolonged starvation for exogenous carbon and nitrogen, was removed by adenosine. This compound also considerably reduced the inhibitory effect of metabolic poisons on the induced synthesis of β-galactosidase. The blockade of induced β-galactosidase synthesis evoked in aerobically grown cells by anaerobic starvation for exogenous sources of carbon and nitrogen was also significantly reduced by adenosine. The weak transient catabolic repression of induced synthesis of β-galactosidase evoked by glucose in non-growing cells ofEscherichia coli deprived of exogenous carbon and nitrogen sources was prevented by adenosine. The total repression caused by higher glucose concentrations was not influenced by this compound. The results are discussed from the point of view of the role of the energy state ofEscherichia coli cells in the regulation of β-galactosidase synthesis.     

Adaptation to nutrient starvation in Rhizobium leguminosarum bv. phaseoli: analysis of survival, stress resistance, and changes in macromolecular synthesis during entry to and exit from stationary phase.

The nitrogen-fixing bacterium Rhizobium leguminosarum bv. phaseoli often has to survive long periods of starvation in the soil, when not in a useful symbiotic relationship with leguminous plants. We report that it can survive carbon, nitrogen, and phosphorus starvation for at least 2 months with little loss of viability. Upon carbon starvation, R. leguminosarum cells were found to undergo reductive cell division. During this period, they acquired the potential for long-term starvation-survival, levels of protein, DNA, and RNA synthesis were decreased to base levels, and pool mRNA was stabilized. The starved cells are ready to rapidly restart growth when nutrients become available. Upon addition of fresh nutrients, there is an immediate increase in the levels of macromolecular synthesis, pool mRNA destabilizes, and the cultures enter exponential growth within 5 to 8 h. The starved cells were cross-protected against pH, heat, osmotic, and oxidative shock. These results provide evidence for a general starvation response in R. leguminosarum similar to that previously found in other bacteria such as Escherichia coli and Vibrio sp.     

Mobilization of sequestered metabolities into degradative reactions by nutritional stress in Neurospora.

The pools of arginine and ornithine rapidly disappear during nitrogen starvation of Neurospora crassa. Much of this disappearance can be accounted for by degradation catalyzed by preexisting catabolic enzymes. Purine degradation is also initiated by nitrogen metabolic stress. Mobilization of these compounds into degradative reactions does not appear to be a general response to nutritional stress since neither carbon starvation nor inhibition of protein synthesis elicits this response. It is suggested that nitrogen starvation may specifically alter the distribution of arginine and ornithine between vesicles and cytosol. This would be sufficient to initiate and maintain their degradation. These result suggest that compartmentation of amino acids provides a metabolic reserve to be utilized during periods of specific nutritional stress.     

Nitrogen-starvation triggers cellular accumulation of triacylglycerol in Metarhizium robertsii

Nitrogen starvation can induce cellular triacylglycerol (TAG) accumulation in different organisms with an unclear mechanism. In this study, we performed nutrient starvation and lipid droplet (LD) proteomics analyses of the filamentous fungus Metarhizium robertsii. Our results indicated that nitrogen starvation activated cell autophagic activity but inhibited the internalization of LDs into vacuoles for degradation. LD proteomic analyses identified an array of differentially accumulated proteins including autophagy-related (ATG) proteins, heat shock proteins, TAG metabolic and phospholipid biosynthetic enzymes when the fungus was grown in different nutrient conditions. In contrast to the highly activated MrATG8, the ATG proteins involved in vacuolar LD internalization were down-regulated after nitrogen starvation. Cellular TAG contents were increased in different ATG-gene null mutants of M. robertsii. In addition, TAG increase could be due to the up-regulation of TAG biogenesis along with the down-regulation of TAG catabolic enzymes in fungal cells after nitrogen deprivation. The data of this study benefit our understanding of the mechanism of nitrogen starvation induced TAG increase in different cells.     

黄孢原毛平革菌突变株抗碳氮营养阻遏产漆酶碳氮生理调控机理

【目的】通过比较不同碳氮营养及其消耗对产漆酶的影响,了解白腐菌模式种黄孢原毛平革菌解除营养阻遏产漆酶代谢的生理生态特性,揭示白腐菌合成漆酶的碳氮生理调控机理。【方法】分别利用限碳限氮(CL-NL)、限碳富氮(CL-NS)、富碳限氮(CS-NL)与富碳富氮(CS-NS)4种条件培养黄孢原毛平革菌野生型(WT)与突变株,比较两者产漆酶动力学、菌体生长、葡萄糖与氨氮消耗差异及其相关性来揭示解除营养阻遏产漆酶调控生理特性,明确C、N营养对产漆酶的生理调控途径。【结果】突变菌株除消耗速率比野生型略慢外,两者氨消耗趋势一致,但对葡萄糖的消耗比野生型快且氨氮浓度对葡萄糖的消耗影响不大。在CL-NL、CL-NS、CS-NL、CS-NS 4种培养条件下,野生型分别在培养后期的第11、14、19和19天的次生代谢时期产生0.107、0.029、12.84和18.05U/L漆酶,启动漆酶合成及酶峰值出现的时间与基质中葡萄糖耗尽或接近耗尽的时刻,或同氨氮消耗至最低值的时刻相对应;与WT产漆酶特性不同,突变株产漆酶伴随整个培养过程且均有两个产酶高峰,分别在培养的第8、7、12天和12天出现298.83、343.14、271.22、251.49U/L漆酶第一个产酶高峰,在培养的第12、13、19和19天产生257.69、298.78、213.81、216.93U/L漆酶的第二个产酶高峰。碳氮营养对产酶的影响显示:两菌株只要初始碳源浓度相同(限碳或富碳),各自产酶动力学趋势基本一致;相反,即使初始氮源浓度相同但其产酶动力学趋势却不同,说明碳源对黄孢原毛平革菌产漆酶的影响比氮源更为重要。【结论】野生型黄孢原毛平革菌产漆酶受碳或氮饥饿调控,碳、氮各自独立发挥作用且在不同的营养条件下由不同营养素所调控,如在限碳条件下产漆酶主要由葡萄糖饥饿启动,而在富碳条件下则由氨氮饥饿所激发,以碳或氮菌体负荷表示是否达到启动酶合成的调控阀值比单纯碳或氮浓度更为合理。突变菌株漆酶合成的启动不受碳、氮营养所阻遏,可能涉及一个全局调控的改变,解除了漆酶合成的营养阻碍。     

An iTRAQ-based quantitative analysis to elaborate the proteomic response of Nostoc sp. PCC 7120 under N2 fixing conditions

Nostoc sp. PCC 7120 is an oxygen-evolving photoautotrophic N2 fixing filamentous cyanobacterium. Upon nitrogen starvation, a range of processes are initiated, such as differentiation of the heterocysts, specific cells where N2 fixation takes place. We have characterized and quantified the proteome of the Nostoc sp. PCC 7120 wild-type strain grown under N2 fixing and non-N2 fixing conditions. To assess global proteome changes in response to environmental changes, measurements were made using the quantitative proteomics tool, iTRAQ, on a whole cell digest. From this approach, a total of 486 different proteins was accurately identified across 2 biological replicate experiments, where 226 identifications contained 2 or more distinct peptides. Results of metabolic regulation will be discussed to demonstrate that proteomics represents an important tool for the development of heterocystous cyanobacteria for future biological H2 production.     

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

The green alga Hematococcus pluvialis accumulates large amounts of the antioxidant astaxanthin under inductive stress conditions, such as nitrogen starvation. The response to nitrogen starvation and high light leads to the accumulation of carbohydrates and fatty acids as well as increased activity of the tricarboxylic acid cycle. Although the behavior of individual pathways has been well investigated, little is known about the systemic effects of the stress response mechanism. Here we present time-resolved metabolite, enzyme activity, and physiological data that capture the metabolic response of H. pluvialis under nitrogen starvation and high light. The data were integrated into a putative genome-scale model of the green alga to in silico test hypotheses of underlying carbon partitioning. The model-based hypothesis testing reinforces the involvement of starch degradation to support fatty acid synthesis in the later stages of the stress response. In addition, our findings support a possible mechanism for the involvement of the increased activity of the tricarboxylic acid cycle in carbon repartitioning. Finally, the in vitro experiments and the in silico modeling presented here emphasize the predictive power of large scale integrative approaches to pinpoint metabolic adjustment to changing environments.     

Characterization of pII family (GlnK1, GlnK2, and GlnB) protein uridylylation in response to nitrogen availability for Rhodopseudomonas palustris

The GlnK and GlnB proteins are members of the pII signal transduction protein family, which is essential in nitrogen regulation due to this protein family's ability to sense internal cellular ammonium levels and control cellular response. The role of GlnK in nitrogen regulation has been studied in a variety of bacteria but previously has been uncharacterized in the purple nonsulfur anoxygenic phototropic bacterium Rhodopseudomonas palustris. R. palustris has tremendous metabolic versatility in its modes of energy generation and carbon metabolism, and it employs a sensitive nitrogen-ammonium regulation system that may vary from that of other commonly studied bacteria. In R. palustris, there are three annotated forms of pII proteins: GlnK1, GlnK2, and GlnB. Here we describe, for the first time, the characterization of GlnK1, GlnK2, and GlnB modifications as a response to nitrogen availability, thereby providing information about how this bacterium regulates the AmtB ammonium transporter and glutamine synthetase, which controls the rate of glutamate to glutamine conversion. Using a strategy of creating C-terminally tagged GlnK and GlnB proteins followed by tandem affinity purification in combination with top-down mass spectrometry, four isoforms of the GlnK2 and GlnB proteins and two isoforms of the GlnK1 protein were characterized at high resolution and mass accuracy. Wild-type or endogenous expression of all three proteins was also examined under normal ammonium conditions and ammonium starvation to ensure that the tagging and affinity purification methods employed did not alter the natural state of the proteins. All three proteins were found to undergo uridylylation under ammonium starvation conditions, presumably to regulate the AmtB ammonium transporter and glutamine synthetase. Under high-ammonium conditions, the GlnK1, GlnK2, and GlnB proteins are unmodified. This experimental protocol involving high-resolution mass spectrometry measurements of intact proteins provides a powerful method of examining the posttranslational modifications that play a crucial role in both the regulation of the AmtB ammonium transporter and glutamine synthetase within R. palustris.     

Effects of liver damage on ketone-body production and nitrogen balance in starved rats

The metabolic effects of intraperitoneal administration of carbon tetrachloride (1ml/kg) were studied in starved rats. The most notable change in circulating substrates was an 80% fall in ketone-body concentrations, which was associated with the doubling of urinary nitrogen losses. The results demonstrate the importance of starvation ketosis in permitting fat mobilization to decrease effectively protein losses during starvation.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.     

Survival of mycobacteria depends on proteasome‐mediated amino acid recycling under nutrient limitation

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S‐proteasome, bacterial prokaryotic ubiquitin‐like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup‐proteasome system (PPS) is important for virulence, yet its physiological role in non‐pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto‐regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.