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Effect of carbon starvation on p-nitrophenol degradation by a Moraxella strain in buffer and river water.

This study examines the effect of carbon starvation on the ability of a Moraxella sp. strain to degrade p-nitrophenol (PNP). Carbon starvation for 24 h decreased the induction time for p-nitrophenol degradation by the bacterium in a minimal salt medium from 6 to 1 h but it did not completely eliminate the induction time. Moraxella cells with 2-day carbon starvation had an induction time of 3 h and the induction time of the 3-day starved cells was 6 h. A 100% increase in density of the non-starved cells did not affect the induction time for p-nitrophenol degradation by the bacterium, indicating that the initial increase in cell density of the carbon-starved culture did not cause the faster onset of p-nitrophenol degradation. However, the initial uptake of p-nitrophenol of the 1-day carbon-starved Moraxella cells was 3-fold higher than the non-starved cells. A green fluorescent protein gene (gfp)-labelled Moraxella (M6 strain) was constructed to examine the survival of and p-nitrophenol degradation by the bacterium in non-sterile river water samples. Similar p-nitrophenol degradation behaviour was observed in the river water samples inoculated with the M6 cells. The time needed for complete degradation of p-nitrophenol by the non-starved M6 was 19-27 and 33 h in samples spiked with 80, 200 and 360 microM p-nitrophenol, respectively. However, the 1-day carbon-starved inocula required about 16 h to degrade the p-nitrophenol completely regardless of its concentration in the water samples. Survival of the carbon-starved and non-starved M6 was not significantly different from each other in the river water regardless of the p-nitrophenol concentration. In the absence of p-nitrophenol, the inoculum density decreased continuously. At 200 and 360 microM p-nitrophenol, the cell densities of M6 increased in the first two days of incubation and declined steadily afterward.

Buffers↗

Iron starvation leads to increased expression of Cu/Zn-superoxide dismutase in Aspergillus.

In a search for iron-regulated proteins of Aspergillus nidulans and Aspergillus fumigatus a 16-kDa protein was identified which is about 5-fold upregulated during iron starvation in both species and which can be approximately 500-fold enriched by simple one-step chromatography on Amberlite XAD-16 resin. N-terminal protein sequence analysis and cloning of the respective A. nidulans cDNA identified this protein as a Cu/Zn-superoxide dismutase (SODA). Northern analysis revealed that upregulation of sodA expression occurs at the level of transcript accumulation. This seems to be a specific low iron response and not a general starvation answer since sodA transcript levels do not respond to carbon or nitrogen starvation. In contrast, copper depletion leads to transcriptional downregulation of sodA. Furthermore, sodA expression was found still to be subject to iron regulation in an A. nidulans mutant lacking SREA, a regulator of iron homeostasis, indicating that sodA expression is regulated by an SREA-independent mechanism. The data presented suggest that SODA plays a protective role under iron deplete conditions.

Amino Acid Sequence↗

Changes in free and conjugated polyamines during starvation of sugarcane juices as analyzed by high-performance liquid chromatography.

Changes of the polyamines (PAs) titer in high- and mid-molecular-mass carbohydrates (HMMCs and MMMCs, respectively) obtained from sugarcane juices stored for 72 h at pH 5.2 or clarified at pH 8.0 have been studied. Cadaverine (CAD) is the most abundant free (S) PA in the MMMC fraction from juices at pH 5.2, whereas putrescine (PUT) was revealed as the main PA at pH 8.0. A slight increase in the free PUT titer can be noted at pH 5.2 for 72 h of juice starvation. PAs from MMMC were mainly conjugated to acid-insoluble (PH) molecules. Accumulation of PH-PAs with the time of starvation was especially significant for PUT and CAD. However, CAD has also been detected in the acid-soluble (SH) fraction and its concentration increases with the time of starvation at pH 5.2. The accumulation pattern of free and conjugated PAs from HMMCs is similar to that found for MMMCs although some differences can be observed. For instance, the increase in free PUT with the time at pH 8.0 was 2.7-times higher in the HMMC fraction than in the MMMC fraction. Conjugated PAs associated to acid-soluble macromolecules (SH fraction) achieved a level in HMMC fractions higher than that observed in the MMMC fraction. Moreover, the reported increase with time that was observed in PH-CAD from the MMMC fraction was not observed in the HMMC fraction, and, finally, the increase in PH-PUT with the time was lower for the HMMC fraction than for the MMMC fraction.

Chromatography, High Pressure Liquid↗

Effects of severe CO(2) starvation on the photosynthetic electron transport chain in tobacco plants.

Tobacco plants (Nicotiana tabacum) were kept in CO(2) free air for several days to investigate the effect of lack of electron acceptors on the photosynthetic electron transport chain. CO(2) starvation resulted in a dramatic decrease in photosynthetic activity. Measurements of the electron transport activity in thylakoid membranes showed that a loss of Photosystem II activity was mainly responsible for the observed decrease in photosynthetic activity. In the absence of CO(2) the plastoquinone pool and the acceptor side of Photosystem I were highly reduced in the dark as shown by far-red light effects on chlorophyll fluorescence and P700 absorption measurements. Reduction of the oxygen content of the CO(2) free air retarded photoinhibitory loss of photosynthetic activity and pigment degradation. Electron flow to oxygen seemed not to be able to counteract the stress induced by severe CO(2) starvation. The data are discussed in terms of a donation of reducing equivalents from mitochondria to chloroplasts and a reduction of the plastoquinone pool via the NAD(P)H-plastoquinone oxidoreductase during CO(2) starvation.

Journal Article↗

Growth of methionine-dependent human prostate cancer (PC-3) is inhibited by ethionine combined with methionine starvation.

Methionine (MET) is required for cell metabolism. MET endogenously synthesized from homocysteine (HCY) supports the proliferation of normal cells, but not that of numerous malignant cells, as shown previously. MET starvation should have an anti-tumour effect, and its deleterious effects on the hosts might be prevented by HCY. Anti-tumour effects of MET starvation must be reinforced by ethionine (ETH), a MET analogue. MET dependency of PC-3, a human prostate cancer cell line, was studied in vitro. Proliferation of PC-3 cells, cultivated in MET-free medium, was 29% compared with growth in MET+HCY- medium. Addition of HCY to MET-free medium increased the proliferation rate to 56%. The concentration of ETH required to decrease the PC-3 cell proliferation rate to 50% (IC50) was 0.5 mg ml(-1) in MET-HCY- medium. ETH-induced inhibition was abolished by MET addition and was reinforced by HCY. PC-3 cell cycle was blocked in the S-G2-phase after 30 h culture in the absence of MET; this blockage was not reversed by addition of HCY. ETH at the IC50 in MET-HCY+ medium blocked DNA replication. Apoptotic cells appeared after 30 h incubation in MET-HCY+ medium only when ETH was added. ATP pools were decreased after 15 h of culture in MET-free medium. In vivo, MET starvation was obtained by feeding tumour-bearing mice a diet containing a synthetic amino acid mixture as the protein supply, in which HCY replaced MET. Given to nude mice bearing xenografted PC-3, from day 1 after grafting and for 3 weeks, this diet inhibited tumour growth (34% on day 20, P < 0.007); this effect was potentiated by ETH (200 mg kg(-1) day(-1) i.p.) (56% on day 20, P < 5 x 10(-5)). The differences between the effects of these two treatments were significant (P < 0.017) and optimal on day 20. These data showed that combination of ETH and HCY slowed the proliferation of prostate cancer cells in vitro and in vivo, decreased ATP synthesis and caused cell cycle arrest and apoptosis. Experimental therapy based on cancer cell MET metabolism deficiency could be efficient for treating advanced prostate cancers refractory to current therapies.

Adenosine Triphosphate↗

A dual role for PP1 in shaping the Msn2-dependent transcriptional response to glucose starvation.

In yeast, glucose depletion elicits a quick response in the transcription of stress-related genes. The main transcriptional activator that orchestrates this response is Msn2, whose nuclear localization and DNA binding are negatively controlled by the cAMP-dependent protein kinase (PKA). Msn2 activation by sudden glucose depletion correlates with a fast but transient decrease in phosphorylation of several sites in its nuclear localization signal (NLS). Here we show that protein phosphatase 1 (PP1) is the direct antagonist of PKA-dependent phosphorylation at the Msn2 nuclear import domain and therefore a potential mediator of glucose starvation signals that target this transcription factor. Apart from PKA, the protein kinase Snf1 can also directly modify one of the Msn2 phosphorylation sites (S582) and thereby repress Msn2 function. Consequently, in snf1 mutants, rephosphorylation of the NLS happens to be much slower during prolonged starvation. Thus, a second, Reg1-dependent form of PP1 indirectly influences Msn2 functionality by modulating Snf1 kinase activation and repression. Different activities of PP1 are therefore involved in shaping induction and adaptation of the transcriptional stress response during acute glucose starvation.

Adaptation, Physiological↗

Starvation-induced expression of autophagy-related genes in Arabidopsis.

BACKGROUND INFORMATION: Autophagy is a catabolic process for degradation of cytoplasmic components in the vacuolar apparatus. A genome-wide survey recently showed evolutionary conservation among autophagy genes in yeast, mammals and plants. To elucidate the molecular and subcellular machinery responsible for the sequestration and subsequent digestion of intracellular material in plants, we utilized a combination of morphological and molecular methods (confocal laser-scanning microscopy, transmission electron microscopy and real-time PCR respectively). RESULTS: Autophagy in Arabidopsis thaliana suspension-cultured cells was induced by carbon starvation, which triggered an immediate arrest of cell growth together with a rapid degradation of cellular proteins. We followed the onset of these responses and, in this report, provide a clear functional classification for the highly polymorphic autophagosomes by which the cell sequesters and degrades a portion of its own cytoplasm. Quantification of autophagy-related structures shows that cells respond to the stress signal by a rapid and massive, but transient burst of autophagic activity, which adapts to the stress signal. We also monitored the real-time expressions of AtATG3, AtATG4a, AtATG4b, AtATG7 and AtATG8a-AtATG8i genes, which are orthologues of yeast genes involved in the Atg8 ubiquitination-like conjugation pathway and are linked to autophagosome formation. We show that these autophagy-related genes are transiently up-regulated in a co-ordinated manner at the onset of starvation. CONCLUSIONS: Sucrose starvation induces autophagy and up-regulates orthologues of the yeast Atg8 conjugation pathway genes in Arabidopsis cultured cells. The AtATG3, AtATG4a, AtATG4b, AtATG7 and AtATG8a-AtATG8i genes are expressed in successive waves that parallel the biochemical and cytological remodelling that takes place. These genes thus serve as early markers for autophagy in plants.

Arabidopsis↗

Synthesis of ribonucleic acid in purine-deficient Escherichia coli and a comparison with the effects of amino acid starvation.

1. Experiments with rifampicin and stringent strains of Escherichia coli (pro(-)purB(-)rel(+)) indicate that purine deficiency does not decrease and may considerably increase the potential for RNA synthesis by RNA polymerase molecules that are bound to DNA and have already commenced transcription. 2. DNA-RNA hybridization experiments indicate that purine starvation increases the distribution of bound RNA polymerase molecules between the cistrons for mRNA and those for stable RNA. 3. Synthesis of beta-galactosidase mRNA is more dependent on the ability to synthesize guanine nucleotides than on the ability to synthesize adenine nucleotides. 4. Amino acid starvation tends to decrease the potential for RNA synthesis by RNA polymerase molecules bound to DNA. 5. Since this effect differs from that due to purine starvation, amino acid control of RNA synthesis does not appear to operate solely by causing a deficiency of purine nucleotides. 6. The results are discussed in terms of the ability to initiate RNA chains and to extend them under different circumstances.

Adenine Nucleotides↗

The biosynthesis of plasmodial myosin during starvation of Physarum polycephalum.

The actomyosin protein complex of Physarum polycephalum was prepared from vegetative and starved plasmodia. The yield of actomyosin per unit wet wt. was the same from both types of plasmodia. Myosin was resolved from the complex by gel filtration and purified by ion-exchange chromatography. The Ca(2+)-stimulated adenosine triphosphatase activities of myosin preparations from vegetative and starved plasmodia were not appreciably different. Synthesis of myosin de novo was shown to occur during the starvation phase of the life-cycle by the isolation of labelled myosin preparations from plasmodia starved in the presence of [2-(14)C]glycine. Fractionation of polyacrylamide gels after gel filtration of labelled myosin confirmed the presence of label in the adenosine triphosphatase-active myosin band. It is concluded that during starvation myosin synthesis continues although there is a net loss of approx. 50% of the total protein. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis of Physarum myosin showed the presence of low-molecular-weight components of the molecule, similar to those of muscle myosins. The content and composition of the free amino acid pool of Physarum was measured at various time-intervals during the vegetative and starvation phases of the life-cycle.

Actomyosin↗

Effects of inositol starvation on the levels of inositol phosphates and inositol lipids in Neurospora crassa.

An inositol-requiring strain of Neurospora crassa was labelled during growth in liquid medium with [3H]inositol, and the levels of inositol phosphates and phosphoinositides were determined under inositol-sufficient and inositol-starved conditions. Because the mutant has an absolute requirement for inositol, the total mass of inositol-containing compounds could be determined. Inositol-containing lipids were identified by deacylation and co-migration with standards on h.p.l.c.; PtdIns3P, PtdIns4P, and PtdIns(4,5)P2 were found in approximately equal amounts, in addition to large amounts of PtdIns. Inositol starvation decreased the level of PtdIns to 10% of the sufficient level, and decreased the levels of the other phosphoinositides to about 25%. A number of inositol phosphates were found, including several InsP3s, InsP4s and InsP5s and phytic acid. Ins(1,4,5)P3 was identified by co-migration with standards on h.p.l.c. and by digestion with inositol phosphomonoesterase. High concentrations of all inositol phosphates were found in the extracellular medium in inositol-starved cultures. Inositol starvation on both liquid and solid agar media decreased the intracellular levels of some inositol phosphates, but increased the levels of phytic acid and several other inositol phosphates which may be its precursors and/or breakdown products. These results may indicate that inositol starvation induces phytic acid synthesis as a protection against the free-radical production and lipid peroxidation characteristic of inositol-less death.

Chromatography, High Pressure Liquid↗

Purification and biochemical characterization of a vacuolar serine endopeptidase induced by glucose starvation in maize roots.

An endopeptidase (designated RSIP, for root-starvation-induced protease) was purified to homogeneity from glucose-starved maize roots. The molecular mass of the enzyme was 59 kDa by SDS/PAGE under reducing conditions and 62 kDa by gel filtration on a Sephacryl S-200 column. The isoelectric point of RSIP was 4.55. The purified enzyme was stable, with no auto-proteolytic activity. The enzyme activity was strongly inhibited by proteinaceous trypsin inhibitors, di-isopropylfluorophosphate, 3,4-dichloroisocoumarin and PMSF, suggesting that the enzyme is a serine protease. The maximum proteolytic activity against different protein substrates occurred at pH 6.5. With the exception of succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin, no hydrolysis was detected with synthetic tryptic, chymotryptic or peptidylglutamate substrates. The determination of the cleavage sites in the oxidized B-Chain of insulin showed specificity for hydrophobic residues at the P2 and P3 positions, indicating that RSIP is distinct from other previously characterized maize endopeptidases. Both subcellular fractionation and immuno-detection in situ indicated that RSIP is localized in the vacuole of the root cells. RSIP is the first vacuolar serine endopeptidase to be identified. Glucose starvation induced RSIP: after 4 days of starvation, RSIP was estimated to constitute 80% of total endopeptidase activity in the root tip. These results suggest that RSIP is implicated in vacuolar autophagic processes triggered by carbon limitation.

Amino Acid Sequence↗

Morphological changes (including filamentation) in Escherichia coli grown under starvation conditions on silicon wafers and other surfaces.

Using a scanning electron microscope, pleomorphism (notably filamentation) was seen when Escherichia coli was grown under starvation conditions for 14 d on microporous silicon wafers, titanium, glass and plastic discs. Under these conditions, the 'standard', rod shaped cell (1-3 microns) failed to separate after division and filaments developed, some as long as 50 microns, with many showing bulbous tips. Filamentation began to occur 5 d after the imposition of starvation conditions. Dumbbell shaped cells were also observed, although apparent 'Y' and 'V'-shaped cells proved to be artefacts, caused by overlapping rods. The implications of the appearance of pleomorphism in E. coli, when grown under starvation conditions, is discussed in relation to its pathogenicity and growth in the environment.

Escherichia coli↗

Starvation-induced Mucts62-mediated coding sequence fusion: a role for ClpXP, Lon, RpoS and Crp.

The formation of araB-lacZ coding sequence fusions in Escherichia coli is a particular type of chromosomal rearrangement induced by Mucts62, a thermoinducible mutant of mutator phage Mu. Fusion formation is controlled by the host physiology. It only occurs after aerobic carbon starvation and requires the phage-encoded transposase pA, suggesting that these growth conditions trigger induction of the Mucts62 prophage. Here, we show that thermal induction of the prophage accelerated araB-lacZ fusion formation, confirming that derepression is a rate-limiting step in the fusion process. Nonetheless, starvation conditions remained essential to complete fusions, suggesting additional levels of physiological regulation. Using a transcriptional fusion indicator system in which the Mu early lytic promoter is fused to the reporter E. coli lacZ gene, we confirmed that the Mucts62 prophage was derepressed in stationary phase (S derepression) at low temperature. S derepression did not apply to prophages that expressed the Mu wild-type repressor. It depended upon the host ClpXP and Lon ATP-dependent proteases and the RpoS stationary phase-specific sigma factor, but not upon Crp. None of these four functions was required for thermal induction. Crp was required for fusion formation, but only when the Mucts62 prophage encoded the transposition/replication activating protein pB. Finally, we found that thermally induced cultures did not return to the repressed state when shifted back to low temperature and, hence, remained activated for accelerated fusion formation upon starvation. The maintenance of the derepressed state required the ClpXP and Lon host proteases and the prophage Ner-regulatory protein. These observations illustrate how the cts62 mutation in Mu repressor provides the prophage with a new way to respond to growth phase-specific regulatory signals and endows the host cell with a new potential for adaptation through the controlled use of the phage transposition machinery.

ATP-Dependent Proteases↗

Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling.

The search for new TB drugs that rapidly and effectively sterilize the tissues and are thus able to shorten the duration of chemotherapy from the current 6 months has been hampered by a lack of understanding of the metabolism of the bacterium when in a 'persistent' or latent form. Little is known about the condition in which the bacilli survive, although laboratory models have shown that Mycobacterium tuberculosis can exist in a non-growing, drug-resistant state that may mimic persistence in vivo. Using nutrient starvation, we have established a model in which M. tuberculosis arrests growth, decreases its respiration rate and is resistant to isoniazid, rifampicin and metronidazole. We have used microarray and proteome analysis to investigate the response of M. tuberculosis to nutrient starvation. Proteome analysis of 6-week-starved cultures revealed the induction of several proteins. Microarray analysis enabled us to monitor gene expression during adaptation to nutrient starvation and confirmed the changes seen at the protein level. This has provided evidence for slowdown of the transcription apparatus, energy metabolism, lipid biosynthesis and cell division in addition to induction of the stringent response and several other genes that may play a role in maintaining long-term survival within the host. Thus, we have generated a model with which we can search for agents active against persistent M. tuberculosis and revealed a number of potential targets expressed under these conditions.

Adaptation, Physiological↗

Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon.

The Bacillus subtilis ferric uptake repressor (Fur) protein coordinates a global transcriptional response to iron starvation. We have used DNA microarrays to define the Fur regulon and the iron starvation stimulon. We identify 20 operons (containing 39 genes) that are derepressed both by mutation of fur and by treatment of cells with the iron chelator 2,2'-dipyridyl. These operons are direct targets of Fur regulation as judged by DNase I footprinting. Analyses of lacZ reporter fusions to six Fur-regulated promoter regions reveal that repression is highly selective for iron. In addition to the Fur regulon, iron starvation induces members of the PerR regulon and leads to reduced expression of cytochromes. However, we did not find any evidence for genes that are directly activated by Fur or repressed by Fur under iron-limiting conditions. Although genome searches using the 19 bp Fur box consensus are useful in identifying candidate Fur-regulated genes, some genes associated with Fur boxes are not demonstrably regulated by Fur, whereas other genes are regulated from sites with little apparent similarity to the conventional Fur consensus.

2,2'-Dipyridyl↗

The effect of starvation stress on the porin protein expression of Escherichia coli in lake water.

AIMS: The aim was to identify changes in outer membrane proteins (omps), OmpA, OmpC and OmpF, in Escherichia coli under starvation conditions in lake water microcosms studied at different temperatures. METHODS AND RESULTS: Escherichia coli was incubated in lake water microcosms at a variety of temperatures and the omps studied using quantitative densitometric analysis of protein bands of sodium dodecyl sulphate gels of omp preparations. The amount of OmpF increased over the incubation period relative to that of OmpC whereas the relative abundance of OmpA declined, most notably at 25 and 37 degrees C. This change was linked to changes in peak cell volume as determined by cell measurements. CONCLUSIONS: Major changes to the omps of E. coli accompany the adaptation of the organism to starvation conditions in lake water microcosms. SIGNIFICANCE AND IMPACT OF THE STUDY: Prolonged starvation affects the relative amounts of outer membrane porins. This study furthers the understanding of the role played by changes in the omp composition in E. coli during survival in lake water environments.

Bacterial Outer Membrane Proteins↗

Increase of lipogenic enzyme mRNA levels in rat white adipose tissue after multiple cycles of starvation-refeeding.

Recently, we have found that despite the significant reduction of body weight after multiple starvation-refeeding cycles, white adipose tissue (WAT) exhibits surprisingly high rates of lipogenesis and lipogenic enzyme activities. The purpose of this study was to determine the response of WAT lipogenic enzyme mRNAs of rats subjected to multiple cycles of 3 days fasting and 3 days of refeeding. Despite the body weight reduction, significant increase of lipogenic enzymes (ie, fatty acid synthase [FAS], acetyl-coenzyme A [CoA] carboxylase [ACC], adenosine triphosphate (ATP)-citrate lyase [ACL], NADP-linked malic enzyme [ME], and glucose 6-phosphate dehydrogenase [G6PDH]) mRNAs in WAT was found after multiple cycles of starvation-refeeding of rats on standard laboratory diet. These findings, together with the results published recently, indicate that multiple cycles of starvation-refeeding cause the increased lipogenesis in WAT by upregulation of the lipogenic enzymes gene expression.

Adipose Tissue↗

Elongation factor Tu.guanosine 3'-diphosphate 5'-diphosphate complex increases the fidelity of proofreading in protein biosynthesis: mechanism for reducing translational errors introduced by amino acid starvation.

Complexes of elongation factor Tu (EF-Tu) with guanosine 3'-diphosphate 5'-diphosphate (ppGpp) bind to ribosomes where they slow the incorporation of aminoacyl-tRNAs into protein by inhibiting both the binding of aminoacyl-tRNA.EF-Tu.GTP ternary complexes and the formation of peptide bonds. The latter action increases the time available for aminoacyl-tRNA rejection by the ribosome and, therefore, increases the effectiveness of proofreading. Synthesis of ppGpp and the formation of EF-Tu.ppGpp occur in vivo in response to amino acid starvation. Our finding, therefore, suggests an explanation for the otherwise puzzling observation that amino acid starvation has, at most, a moderate effect on the fidelity of protein synthesis in wild-type Escherichia coli. We suggest that an EF-Tu.ppGpp-induced increase in the effectiveness of proofreading buffers the overall translational fidelity of these cells against amino acid starvation-induced errors in initial selection of aminoacyl-tRNA ternary complexes.

Escherichia coli↗