The effect of amino acids on growth and phosphate metabolism in a prototrophic yeast strain

Ludwig, J R; Oliver, Stephen G.; McLaughlin, Calvin S.

doi:10.1016/0006-291x(77)90054-7

Cited by 32 publications

(15 citation statements)

References 8 publications

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“…The discrepancy may result from the presence of longer chain polyphosphate intermediates, which are not resolved by the acid extraction, but show up in the NMR spectrum. Previous reports suggest that in yeast, amines and basic amino acids also induced hydrolysis of polyphosphates and accumulation of tripolyphosphate (6,13,14). These observations suggest that hydrolysis of long-chain polyphosphates to tripolyphosphate may provide a general protective mechanism to counterbalance alkaline stress and to accumulate amines in different classes of microorganisms.…”

Section: Discussionmentioning

confidence: 76%

Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina

Pick

Weiss²

1991

Plant Physiol.

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The location and mobilization of polyphosphates in response to an amine-induced alkaline stress were studied in the halotolerant alga Dunaliella salina. The following observations suggest that polyphosphates accumulate in acidic vacuoles: (a) Accumulation of large amounts of polyphosphates is manifested as intravacuolar dense osmiophilic bodies in electron micrographs. (b) Uptake of amines into the vacuoles induces massive hydrolysis of polyphosphates, demonstrated by in vivo 31P-nuclear magnetic resonance, and by analysis of hydrolytic products on thin layer chromatograms. The analysis indicates that: (a) Polyphosphate hydrolysis is kinetically correlated with amine accumulation and with the recovery of cytoplasmic pH. (b) The major hydrolytic product is tripolyphosphate. (c) The peak position of the tripolyphosphate terminal phosphate in nuclear magnetic resonance spectra is progressively shifted as the cells recover, indicating that the pH inside the vacuoles increases while the pH in the cytoplasm decreases. (d) In lysed cell preparations, in which vacuoles become exposed to the external pH, mild alkalinization in the absence of amines induces polyphosphate hydrolysis to tripolyphosphates. It is suggested that amine accumulation within vacuoles activates a specific phosphatase, which hydrolyzes long-chain polyphosphates to tripolyphosphates. The hydrolysis increases the capacity of the vacuoles to sequester amines from the cytoplasm probably by releasing protons required to buffer the amine, and leads to recovery of cytoplasmic pH. Thus, polyphosphate hydrolysis provides a high-capacity buffering system that sustains amine compartmentation into vacuoles and protects cytoplasmic pH. (2). In a preliminary recent work, we have demonstrated that ammonia at alkaline pH induces alkalinization of the cells and leads to massive hydrolysis of polyphosphates, which is manifested in 31P-NMR spectra and by accumulation of large amounts of tripolyphosphate in the cells (17). The correlation between polyphosphate hydrolysis and recovery of cytoplasmic pH suggested a possible connection to pH homeostasis in this alga. However, this work did not address the location of polyphosphates in the cell, the mechanism of recovery from alkaline stress, and whether the response of Dunaliella is specific to ammonia. In the preceding work, we show that ammonia as well as other amines accumulate in acidic vacuoles in Dunaliella and that cytoplasmic pH homeostasis involves the compartmentations of amines into the vacuoles (18). In this work, we demonstrate that polyphosphates seem to be located in the same vacuoles and that various amines induce their hydrolysis as a consequence of intracellular pH changes. MATERIALS AND METHODS NMR MeasurementsDunaliella salina cells were grown in batch cultures within an illuminated New Brunswick Psychotherm incubator in 0.5 M NaCl medium as previously described (2). For in vivo NMR measurements, cells were trapped in 3% agarose beads at a concentration of 6 to 8 x 108 cells/mL and perfused...

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Section: Discussionmentioning

confidence: 76%

Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina

Pick

Weiss²

1991

Plant Physiol.

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“…Instead, it could contain short-chain polyphosphates, which have been shown to accumulate in, for example, Halobacterium volcanii when entering the stationary phase (37). Similar phosphate compounds have been characterized in Saccharomyces cerevisiae (25,38) and are apparently ubiquitous in nature (23). In particular, in E. coli, inorganic polyphosphates synthesized by polyphosphate kinase mediate the resistance of the bacteria to stressful agents and prolong the survival of the organism in the stationary phase (32).…”

Section: Discussionmentioning

confidence: 95%

Characterization of the stringent and relaxed responses of Streptococcus equisimilis

Mechold

Malke

1997

J Bacteriol

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The 739-codon rel Seq gene of Streptococcus equisimilis H46A is bifunctional, encoding a strong guanosine 3,5-bis(diphosphate) 3-pyrophosphohydrolase (ppGppase) and a weaker ribosome-independent ATP:GTP 3-pyrophosphoryltransferase [(p)ppGpp synthetase]. To analyze the function of this gene, (p)ppGpp accumulation patterns as well as protein and RNA synthesis were compared during amino acid deprivation and glucose exhaustion between the wild type and an insertion mutant carrying a rel Seq gene disrupted at codon 216. We found that under normal conditions, both strains contained basal levels of (p)ppGpp. Amino acid deprivation imposed by pseudomonic acid or isoleucine hydroxamate triggered a rel Seq -dependent stringent response characterized by rapid (p)ppGpp accumulation at the expense of GTP and abrupt cessation of net RNA accumulation in the wild type but not in the mutant. Tetracycline added to block (p)ppGpp synthesis caused the accumulated (p)ppGpp to degrade rapidly, with a concomitant increase of the GTP pool (decay constant of ppGpp, Ϸ0.7 min ؊1). Simultaneous addition of pseudomonic acid and tetracycline to mimic a relaxed response caused wild-type RNA synthesis to proceed at rates approximating those seen under either condition in the mutant. Glucose exhaustion provoked the (p)ppGpp accumulation response in both the wild type and the rel Seq insertion mutant, consistent with the block of net RNA accumulation in both strains. Although the source of (p)ppGpp synthesis during glucose exhaustion remains to be determined, these findings reinforce the idea entertained previously that rel Seq fulfils functions that reside separately in the paralogous relA and spoT genes of Escherichia coli. Analysis of (p)ppGpp accumulation patterns was complicated by finding an unknown phosphorylated compound that comigrated with ppGpp under two standard thin-layer chromatography conditions. Unlike ppGpp, this compound did not adsorb to charcoal and did not accumulate appreciably during isoleucine deprivation. Like ppGpp, the unknown compound did accumulate during energy source starvation.Bacteria have evolved elaborate strategies that enable them to adjust their macromolecular syntheses when their nutrient supply becomes limiting or other environmental stresses are imposed. Among the responses involving the modulation of gene expression is the well-known stringent response, the hallmarks of which include the restriction of the synthesis of stable RNAs, the stimulation of certain anabolic reaction pathways, and the induction of stationary-phase-specific genes as results of amino acid limitation or inhibition of tRNA amino acylation. This pleiotropic response is mediated by the accumulation of GDP and GTP derivatives that carry a pyrophosphate group on the 3Ј-hydroxyl position of ribose, i.e., ppGpp and pppGpp, respectively, hereafter together designated (p)ppGpp (see references 3 and 9 for recent reviews). Depending on the nature of the nutritional stress imposed, there are two different sources for (p)ppGpp accumulation in E...

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“…Thus, a number of phosphorylated compounds are produced by H. volcanii at the inception of the late exponential phase of growth or after prolonged nutrient or energy starvation. One hypothesis for this phenomenon is that the molecules produced are short-chain polyphosphates, which have been shown to be produced by other microbial organisms (13,18,20). To verify this hypothesis, formic acid extracts were prepared from S. cerevisiae, an organism with well-characterized polyphosphate expression (13,18), and the spots obtained were compared with those produced by H. volcanii.…”

Section: Resultsmentioning

confidence: 99%

“…One hypothesis for this phenomenon is that the molecules produced are short-chain polyphosphates, which have been shown to be produced by other microbial organisms (13,18,20). To verify this hypothesis, formic acid extracts were prepared from S. cerevisiae, an organism with well-characterized polyphosphate expression (13,18), and the spots obtained were compared with those produced by H. volcanii. Samples obtained from both organisms, from the late exponential and stationary phases of growth, were analyzed by polyethylenimine thinlayer chromatography (Fig.…”

Section: Resultsmentioning

confidence: 99%

Lack of production of (p)ppGpp in Halobacterium volcanii under conditions that are effective in the eubacteria

1995

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The stringent halobacterial strain Haloferax volcanii was subjected to a set of physiological conditions different from amino acid starvation that are known to cause production of guanosine polyphosphates [(p)pp Gpp] in eubacteria via the relA-independent (spoT) pathway. The conditions used were temperature upshift, treatment with cyanide, and total starvation. Under none of these conditions were detectable levels of (p)ppGpp observed. This result, in conjunction with our previous finding that (p)ppGpp synthesis does not occur under amino acid starvation, leads to the conclusion that in halobacteria both growth rate control and stringency are probably governed by mechanisms that operate in the absence of ppGpp. During exponential growth, a low level of phosphorylated compounds with electrophoretic mobilities similar, but not identical, to that of (p)ppGpp were observed. The intracellular concentration of these compounds increased considerably during the stationary phase of growth and with all of the treatments used. The compounds were identified as short-chain polyphosphates identical to those found under similar conditions in Saccharomyces cerevisiae.In 1969, Cashel and Gallant reported that wild-type Escherichia coli is capable of producing two unusual compounds originally named magic spots I and II and later identified as guanosine tetra-and pentaphosphates, respectively [collectively referred to as (p)ppGpp] (3). These guanosine nucleotides are generated in a relA-dependent manner by the relA gene product, (p)ppGpp synthetase I, and in a relA-independent manner by the spoT gene product, the SpoT enzyme, which is also implicated in (p)ppGpp degradation. Kinetic studies have shown that pppGpp is formed from GTP, that ppGpp is derived from pppGpp, and that pppGpp inhibits its own synthesis (12).In eubacteria, the rate of stable RNA synthesis at different growth rates is subject to a control mechanism that is distinct from that of other cellular molecules (growth rate control of RNA synthesis). An inverse correlation between the ppGpp concentration, produced in this case by the relA-independent pathway, and the rate of stable RNA synthesis was observed at a wide range of different growth rates (5), which suggested that ppGpp could act as an effector molecule negatively regulating stable RNA synthesis in growth rate control. The isolation by Xiao et al. (21) of relA spoT mutants unable to produce ppGpp permitted Gaal and Gourse (8) to show that ppGpp either is not the mediator or does not act alone, since growth rate control is normal in the double mutants. Hernandez and Bremer have recently shown that in wild-type E. coli, three factors increase with the growth rate: RNA polymerase concentration, RNA polymerase activity, and the distribution of active RNA polymerase between stable and mRNA genes. In an E. coli⌬relA⌬spoT mutant that does not produce ppGpp, RNA polymerase synthesis and activity increase with the growth rate but the distribution of active RNA polymerase between stable and mRNA genes does not. This...

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The effect of amino acids on growth and phosphate metabolism in a prototrophic yeast strain

Cited by 32 publications

References 8 publications

Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina

Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina

Characterization of the stringent and relaxed responses of Streptococcus equisimilis

Lack of production of (p)ppGpp in Halobacterium volcanii under conditions that are effective in the eubacteria

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