A novel nucleotide implicated in the response of E. coli to energy source downshift

Gallant, Jonathan; Shell, Linda; Bittner, Rex

doi:10.1016/0092-8674(76)90257-9

Cited by 59 publications

(23 citation statements)

References 20 publications

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“…Notably, Ndx8 produced pGp by sequential hydrolysis of two phosphodiester bonds in ppGpp, whereas pGp and two other intermediates could not be detected from the metabolomics screening in this study. For E. coli, previous in vitro studies reported the activity of (p)ppGpp degradation resulting in the production of pGp, ppGp, and pGpp (9), which are regarded as probable sources for the production of guanosine polyphosphate derivatives in vivo (10,55). Moreover, one of such a product, ppGp, is proposed to act as a strong stringent factor inhibiting purine biosynthesis (56,57).…”

Section: Discussionmentioning

confidence: 99%

Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

Ooga

Ohashi

Kuramitsu

et al. 2009

Journal of Biological Chemistry

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A major bacterial alarmone, guanosine 3,5-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3,5-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers.The stringent response, which is a pleiotropic adaptation to nutritional starvation and stress, is broadly conserved in bacteria and plant chloroplasts (1, 2). This physiological control is dominated by a rapid synthesis/degradation cycle of the stringent alarmones guanosine 3Ј-pyrophosphate 5Ј-triphosphate (pppGpp) 4 or guanosine 3Ј,5Ј-bispyrophosphate (ppGpp) ( Fig. 1) (3). In Escherichia coli grown under amino acid depletion, pppGpp is synthesized from ATP and GTP by ribosome-associated RelA activity and subsequently converted to ppGpp by the activity of 5Ј-nucleotidase (4). Besides this pathway, ppGpp is also directly synthesized from GDP by the activity of RelA. Thereafter, ppGpp is degraded to GDP and pyrophosphate by the activity of SpoT, which is a RelA homolog with reciprocal activities for ppGpp degradation and synthesis (5, 6). For several bacteria, SpoT physiologically acts as both the ppGpp synthetase and hydrolase and is known as the Rel/Spo homolog. The metabolism of these alarmone nucleotides is understood based on the function of these two enzymes, whereas recent studies have identified alternative players acting as ppGpp synthetase in bacteria (7,8). On the other hand, for the degradation of ppGpp, previous studies reported that SpoT-independent (p)ppGpp degradation activity exists in bacteria, whereas the biological meaning(s) and enzymatic mechanism(s) of these alternative pathways have not been characterized in any organisms (9 -12).The Nudix hydrolases constitute a large family of proteins that share a highly conserved amino acid sequence, the so-called "Nudix motif," which is distribut...

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Section: Discussionmentioning

confidence: 99%

Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

Ooga

Ohashi

Kuramitsu

et al. 2009

Journal of Biological Chemistry

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“…Ascending chromatography was performed in 1.5 M KH2PO4 at pH 3.4 (6). In two-dimensional chromatography, 1.5 M LiCl and 2 M HCOOH in the first dimension and 1.5 M KH2PO4 (pH 3.4) in the second dimension were used (17). In some cases, results were further verified by using another two-dimensional solvent system, in which the first-dimensional solvent contained 0.75 M Tris, 0.45 M HCl, and 0.5 M LiCl and the second-dimensional solvent contained 74 g of NH4HSO4, 4 g of Na2EDTA, and 100 ml of water (3).…”

Section: Methodsmentioning

confidence: 99%

“…The control of cellular and metabolic processes in bacterial cells can be mediated through a number of regulatory proteins and nucleotides (17,29,36,41). For example, the nucleotide guanosine-3',5'-bis-pyrophosphate (ppGpp) has been implicated in the regulation of a wide array of distinct physiological processes in Escherichia coli and a few other bacteria (8,15,33,39,42).…”

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confidence: 99%

The role of guanosine-3',5'-bis-pyrophosphate in mediating antimicrobial activity of the antibiotic 3,5-dihydroxy-4-ethyl-trans-stilbene

Sundar

Chang

1992

Antimicrob Agents Chemother

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The mode of action of 3,5-dihydroxy-4-ethyl-trans-stilbene (ES), an antibiotic produced by Xenorhabdus luminescens symbiotically associated with an entomopathogenic nematode, was investigated. ES was active against gram-positive and a number of gram-negative bacteria. In susceptible bacteria this antibiotic caused the inhibition of total RNA synthesis and, to a lesser extent, protein synthesis. At or above MICs, ES triggered a substantial accumulation of an intracellular regulatory compound, guanosine-3',5'-bis-pyrophosphate (ppGpp). This response was also noticed in species of bacteria which have previously not been shown to use ppGpp as a regulatory molecule. The involvement of ppGpp in antibiotic action was confirmed by using an isogenic stringent and a relaxed pair of Escherichia coli strains. The fact that the accumulation of ppGpp was correlated with the susceptibility of various gram-positive and gram-negative bacteria to ES suggests that this nucleotide is involved in the regulation of RNA synthesis and growth in all these microorganisms. Thus, inhibition of RNA synthesis via an increase in ppGpp concentrations may represent a mechanism that is prevalent among most bacteria and one that could be exploited for achieving a rapid inhibition of bacterial growth.

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“…Extracts (20 ,ul) were spotted in two 10-,ul applications and blown dry. The first-dimension solvent system (solvent system 2 of Gallant et al [9]: 2 M HCOOH, 1.5 M LiCl [pH 3.4]) was titrated by adding solid NaOH to just less than pH 3.4 and then making the final adjustment with 10 M NaOH. The plates were developed in the first dimension to the top (-4 to 6 h), washed in cold methanol for 20 min, and blown dry (-2 h).…”

Section: Methodsmentioning

confidence: 99%

Correlation between histidine operon expression and guanosine 5'-diphosphate-3'-diphosphate levels during amino acid downshift in stringent and relaxed strains of Salmonella typhimurium

et al. 1989

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We have analyzed the correlation of attenuator-independent expression of the Salmonella typhimurium histidine operon in vivo with levels of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate. Amino acid downshift caused by serine hydroxamate addition increased his expression in a relA+ strain and decreased his expression in a relA mutant, whereas levels of guanosine 5'-diphosphate-3'-diphosphate varied in parallel with the changes in his expression in the two strains. In several experiments, overall variations in his expression ranged from 20-to 60-fold after downshift. The mild downshift allowed growth of the cultures to continue at near-preshift rates. Serine hydroxamate addition was also used to analyze the effect of amino acid downshift on induced expression of wild-type and mutant lac promoters. There was a 12-fold difference in lac expression when a relA+-reLA1 pair was subjected to mild starvation but only a 3-fold difference when the strains carried the lacZpL8UV5 promoter mutation. These results suggest that guanosine 5'-diphosphate-3'-diphosphate stimulates gene expression in vivo at the level of transcription initiation.The histidine operons of Salmonella typhimurium and Escherichia coli are under control of two distinct regulatory mechanisms: (i) a well-characterized mechanism with translational control of transcription termination (attenuation mechanism; reviewed in references 1 and 28); and (ii) a less well-defined mechanism involving the "alarmone" guanosine 5'-diphosphate 3'-diphosphate (ppGpp), which mediates general amino acid control (reviewed in references 1 and 5). Attenuation regulates his expression in response to the levels of charged histidyl-tRNA specifically, whereas ppGpp attunes his expression to the availability of amino acids in general (24) and is the focus of this study.Expression of the his operon has been correlated with ppGpp levels both in vivo (22, 24, 29, 30) and in vitro (21, 24). Correlations in vivo have usually made use of changes in steady-state growth conditions or mutants (spoT) defective in ppGpp degradation, which alter ppGpp levels at most twoto fourfold. Correspondingly, the expression of the his operon varies only two-to fourfold under these conditions. In addition, expression of the his operon is stimulated to nearly maximum levels by the basal ppGpp concentration present in cultures grown in minimal medium (24). Therefore, amino acid downshift of a stringent (relA') strain grown in minimal medium, which causes a large accumulation of ppGpp, does not result in much increase in his expression. In contrast to these results in vivo, attenuatorindependent his expression in vitro has been shown to vary over a 10-to 20-fold range in correlation with ppGpp levels * Corresponding author. (20, 24). These results were obtained with a DNA-dependent in vitro protein-synthesizing system with an S-30 (30,000 x g supernatant) cell extract devoid of ppGpp so that the full extent of stimulation by added ppGpp could be assessed.Determination of the full range of effect of p...

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A novel nucleotide implicated in the response of E. coli to energy source downshift

Cited by 59 publications

References 20 publications

Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

The role of guanosine-3',5'-bis-pyrophosphate in mediating antimicrobial activity of the antibiotic 3,5-dihydroxy-4-ethyl-trans-stilbene

Correlation between histidine operon expression and guanosine 5'-diphosphate-3'-diphosphate levels during amino acid downshift in stringent and relaxed strains of Salmonella typhimurium

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