Characterization of the guanosine 5'-triphosphate 3'-diphosphate and guanosine 5'-diphosphate 3'-diphosphate degradation reaction catalyzed by a specific pyrophosphorylase from Escherichia coli.

Guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) are specifically degraded by a manganese-dependent pyrophosphorylase present in spoT+ but not in spoT- strains of Escherichia coli, indicating that the enzyme is the spoT gene product. The enzyme catalyzes the release of pyrophosphate from the 3' position of ppGpp or pppGpp, yielding ppG and pppG, respectively; pppGpp could not be detected as an intermediate in the decay reaction. Degradation of (p)ppGpp is optimal in the presence of 200 to 300 mM potassium or sodium acetate, at a pH of 7.5 to 8 and a temperature of 37 degrees C.     

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.

The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.     

Nitrofurantoin prompts the stringent response in Bacillus subtilis

Nitrofurantoin causes the stringent response in Bacillus subtilis. After exposure of a stringent strain to this drug, the intracellular concentrations of guanosine 3'-diphosphate 5'-diphosphate (ppGpp), guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and ATP increased, while that of GTP decreased. In a relaxed strain no accumulation of ppGpp or pppGpp was observed, but both GTP and ATP declined after the addition of nitrofurantoin. Protein synthesis was equally sensitive to nitrofurantoin in both the stringent and relaxed strains, but the drug inhibited RNA accumulation only in the stringent strain, not in the relaxed strain. Nitrofurantoin also caused the accumulation of ppGpp in Escherichia coli and Serratia marcescens.     

Synthesis of guanosine tetra- and pentaphosphates by the obligately anaerobic bacterium Bacteroides thetaiotaomicron in response to molecular oxygen.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were detected in formic acid extracts of air-exposed culutres of Bacteroides thetaiotaomicron. The identification of ppGpp and pppGpp in B. thetaiotaomicron was based on the following results: (i) cochromatography of 32P-labeled hyperphosphorylated nucleotides in two different two-dimensional solvent systems with authentic ppGpp and pppGpp; (ii) incorporation of [3H]guanosine into the putative ppGpp and pppGpp; (iii) alkaline lability; and (iv) resistance, to periodate oxidation. There was a marked increase in the concentration of ppGpp and pppGpp after shift from anaerobic to aerobic conditions, and accumulation of both ppGpp and pppGpp was blocked under these conditions by pretreatment of the culture with rifampin or tetracycline. Growth and incorporation of [3H]guanosine, [3H]tymidine, [14C]succinate, and L-[35S]methionine into macromolecules were inhibited immediately upon exposure to air. The accumulation of ppGpp and pppGpp in B. thetaiotaomicron upon exposure to air may represent a novel signal for synthesis of these compounds.     

Intracellular levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) in cultures of Streptomyces griseus producing streptomycin.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were identified in the vegative mycelium of Streptomyces griseus. Adenosine 5'-diphosphate 3'-diphosphate (ppApp) and adenosine 5'-triphosphate 3'-diphosphate (pppApp) were not present but several other phosphorus-containing compounds which may have been inorganic polyphosphates were detected. During exponential growth of S. griseus the concentrations of ppGpp and pppGpp were several times higher than in the stationary stage. They fell sharply when exponential growth ended and then remained at an almost constant basal level. For the tetraphosphate the maximum concentration was about 50, and for the basal level about 10, pmol per millilitre of a culture with an optical density of 1.0. Production of streptomycin started several hours after exponential growth had ended and the concentrations of ppGpp and pppGpp had fallen. Streptomycin synthesis was delayed if the cells were resuspended just before production started in fresh medium lacking phosphate, but it was not delayed by glucose starvation. Both cultures, as well as cultures transferred to nitrogen-free medium, showed an immediate increase in ppGpp content to about four-fold the basal level. The results suggest that the guanosine polyphosphates do not directly control initiation of streptomycin production in S. griseus. Twelve additional species of Streptomyces examined all contained ppGpp and pppGpp.     

Guanosine 5'-triphosphate, 3'-diphosphate 5'-phosphohydrolase. Purification and substrate specificity

The regulatory nucleotide guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) and its precursor guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) are accumulated during stringent response in bacterial cells. The enzyme pppGpp-5'-phosphohydrolase, which catalyzes the conversion of pppGpp to ppGpp, was partially purified from Escherichia coli. It has Mr = 140,000 and an apparent Km of 0.11 mM for pppGpp. It requires Mg2+ and a monovalent cation. NH4+ is preferred over K+, while Na+ is inactive. The enzyme does not hydrolyze GTP, ATP, pppApp, or ppGpp. It is also not effectively inhibited by these nucleotides. pppGpp-5'-phosphohydrolase hydrolyzes the 3'-monophosphate analog pppGp equally well (apparent Km of 0.13 mM), yielding the recently identified MS III nucleotide (ppGp). pppGpp-5'-phosphohydrolase does not have RNA 5'-terminal gamma-phosphatase activity; however, 5'-terminal phosphates are released by pppGpp-5'-phosphohydrolase when the GTP-terminated RNA chains are first converted into oligonucleotides by RNase A treatment. pppGpp-5'-phosphohydrolase was found to actively hydrolyze the dinucleotide fragment pppGpNp but exhibited very low activity toward longer chain fragments. The 3'-unphosphorylated dinucleotide pppGpN was, however, not hydrolyzed. The ability of pppGpp-5'-phosphohydrolase to hydrolyze pppGpp, pppGp, and pppGpNp, but not pppG and pppGpN, indicates that pppGpp-5'-phosphohydrolase is rather nonspecific toward the 3'-OH substitutions of the substrates although a free, unsubstituted phosphate group at the 3'-OH position is essential.     

The regulation of stable RNA synthesis in the blue-green alga Anacystis nidulans: effect of leucine deprivation and 5-methyltryptophan inhibition.

The expression of phenomena associated with the bacterial function controlling RNA synthesis was studied in leucine-deprived or 5-methyltryptophan-treated cultures of Anacystis nidulans. Both procedures retarded cell growth, RNA and protein accumulation, elicited the accumulation of high intracellular concentrations of guanosine 5'-diphosphate-3'-diphosphate(ppGpp)and guanosine5'-triphosphate-3'-diphosphate(pppGpp),and promoted a regime of non-coordinate synthesis of stable and messenger RNA. The rate of polymerization of nascent RNA chains did not appear to be retarded in the growth-limited cultures.     

Promoter- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon.

Expression of the histidine (his) operon in Salmonella typhimurium was found to be positively correlated with the intracellular level of guanosine tetraphosphate (ppGpp). Limitation for amino acids other than histidine elicited a histidine-independent metabolic regulation of the operon. In bacteria grown at decreased growth rates, his operon expression was metabolically regulated up to a point, after which further decreases in growth rate no longer resulted in further enhancement of operon expression. Studies using strains carrying various regulatory and deletion mutations indicated that metabolic regulation is achieved predominantly by increased RNA chain initiations at the primary (P1) and internal (P2) promoters. Metabolic regulation ordinarly did not involve changes in RNA chain terminations at the attenuator site of the his operon. A model is proposed that involves ppGpp-induced changes in RNA polymerase initiation specificity at particular promoters. A second, special form of metabolic regulation may operate which also is histidine independent, but does involve relief of attenuation.     

Synthesis of pppGpp by ribosomes from an Escherichia coli spoT mutant and the metabolic relationship between pppGpp and ppGpp.

Both ribosomes and a cell-free extract (S-30) prepared from an Escherichia coli spoT mutant catalyzed the synthesis of guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) as efficiently as did ribosomes and S-30 from a spoT+ strain. In both cases, the level of pppGpp reached its maximum before ppGpp maximally accumulated. pppGpp added to the ribosome system was rapidly converted to ppGpp. These results indicate that the spoT+ gene product may not have a direct role in the synthesis of pppGpp and that pppGpp is a precursor of ppGpp.     

Guanosine polyphosphate concentration and stable RNA synthesis in Bacillus subtilis following suppression of protein synthesis

Amount of guanosine-5'-triphosphate, 3'-diphosphate (pppGpp) and guanosine-5'-diphosphate, 3'-diphosphate (ppGpp) in the cells of b. subtilis increased several times during starvation for lysine or after treatment with serine hydroxamate (analog of serine) or norvaline (analog of leucine), or in the presence of trimethoprim, which induced deficiency of methionine and leucine. In exponentially growing cells the concentration of pppGpp was found to be 10-20 pmol/A600. When serine hydroxamate or trimethoprim were added, concentration of pppGpp increased to 500-800 pmol/A600 and then slowly diminished. Elimination of lysine or addition to the culture medium of norvaline caused slight transitory accumulation of pppGpp (150 pmol/A600). The amount of another nucleotide ppGpp was always 2-3 times lower than one of pppGpp. Accumulation of (p)ppGpp in rel+ cells was accompanied by cessation of stable RNA synthesis. Under conditions described above rel- cells continued RNA synthesis and did not accumulate (p)ppGpp. In the rel+ cells treated with serine hydroxamate synthesis of stable RNA resumed and the amount of (p)ppGpp decreased after addition of serine or tetracycline and chloramphenicol. The half-life period for pppGpp in the presence of chloramphenicol was determined to be 30-40 seconds. Thus, during aminoacyl-tRNA deficiency rel+ cells of B. subtilis accumulate (p)ppGpp, which are believed to participate in negative regulation of RNA synthesis. Slight accumulation of pppGpp without concomitant inhibition of stable RNA synthesis was observed after treatment of growing cells with chloramphenicol.     

Metabolic regulation of the histidine operon in Escherichia coli and Salmonella typhimurium

Expression of the histidine operon in Escherichia coli cells in contrast to the one in Salmonella typhimurium is changed proportionally to cells growth rate on the different carbon sources. The specific activity of histidinol-dehydrogenase is repressed by addition of 19 amino acids both in Escherichia coli and Salmonella typhimurium independent of the growth medium used. Using of Escherichia coli and Salmonella typhimurium strains containing the heterologous histidine operons made possible to demonstrate the dependence of the histidine operon metabolic regulation to be determined by the operon itself but not by the specificity of the recipient cells. ppGpp was shown to be a positive regulator of the histidine operon expression in Escherichia coli.     

Precise mapping of the gpp gene involved in guanosine tetraphosphate synthesis and ilvC-gpp deletion in the region of the Escherichia coli chromosome

Using the set of transducing lambda phages the gpp gene, responsible for pppGpp to ppGpp conversion, was localized between rep and trxA genes on 85 min of the Escherichia coli genetic map. Taking advantage of the Tn10 transposon inserted into the adjacent ilvY locus, we deleted the region of E. coli chromosome covering ilvC, rep and gpp genes. The metabolism of (p)ppGpp in the deletion-containing cells confirms that the product of the gpp gene, guanosine pentaphosphatase, is not the only enzyme, responsible for pppGpp degradation and ppGpp synthesis.     

Activation of ppGpp-3'-pyrophosphohydrolase by a supernatant factor and ATP

The breakdown of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) into GDP and PPi is catalyzed by a Mn2+-dependent 3'-pyrophosphohydrolase, the translation product of the spoT gene. The escherichia coli enzyme is normally found to be associated with the "crude" ribosome fraction. It is reported here that the guanosine 5'-diphosphate, 3'-diphosphate 3'-pyrophosphohydrolase activity in this fraction is activated by ATP in the presence of a relatively heat-stable, low molecular weight, supernatant factor (BS100). This stimulation is not due to a removal of reaction products such as by the phosphorylation of GDP to GTP or by the hydrolysis of PPi. Hydrolysis of ATP is probably required because neither adenosine 5'-(3-thio)triphosphate nor adenosine 5'-(beta, gamma-imido)triphosphate can substitute for ATP. Levallorphan, a morphine analog, which had been shown to inhibit in vivo ppGpp degradation, inhibits specifically the stimulation of ppGpp hydrolysis by ATP and the supernatant factor. The possible relationship of this system and the in vivo energy-dependent control of ppGpp degradation is discussed.     

Effect of unusual guanosine nucleotides on the activities of some Escherichia coli cellular enzymes

Unusual guanosine nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp, also known as MSI) and guanosine 5'-diphosphate 3'-monophosphate (ppGp, also known as MSIII) accumulate to high concentrations in wild-type cells of Escherichia coli during amino acid starvation. We reported here that both nucleotides strongly inhibit the activity of enzymes IMP dehydrogenase and adenylosuccinate synthetase, the first enzymes of the guanylate and adenylate biosynthetic pathways. In both cases, ppGP (MSII) is a stronger inhibitor than ppGpp (MSI). On the other hand, these two nucleotides exhibited opposite effects on the activity of phosphoenolpyruvate carboxylase, the enzyme that utilizes phosphoenolpyruvate. At their respective physiological concentrations, the activity of phosphoenolpyruvate carboxylase is activated by ppGpp and inhibited by ppGp.     

Evidence for new factors in the coordinate regulation of energy metabolism in Escherichia coli. Effects of hypoxia, chloramphenicol succinate, and 2,4-dinitrophenol on glucose utilization, glycogen synthesis, adenylate energy charge, and hexose phosphates during the first two periods of nitrogen starvation.

We studied the effects of decreased aeration, chloramphenicol succinate, and 2,4-dinitrophenol on the cellular rates of glycogen synthesis and glucose utilization and on the cellular concentrations of adenine nucleotides, glucose 6-phosphate, fructose 1,6-diphosphate, and phosphoenolpyruvate during the first two periods of nitrogen starvation of Escherichia coli W4597(K). A quantitative relationship between the changes in the rates and the accompanying changes in the hexose phosphates is demonstrated. However, the relationship for glycogen synthesis is different in different sets of metabolic conditions. We suggest that this difference reflects a change in the steady state level of a previously unknown effector of ADP-glucose synthetase (glucose 1-phosphate adenylyltransferase, EC 2.7.7.27) the rate-limiting enzyme of bacterial glycogen synthesis. We show that the properties of the hypothetical in vivo effector are consistent with the inhibitory effects of ppGpp (guanosine 3'-diphosphate 5'-diphosphate) and pppGpp (guanosine 3'-diphosphate 5'-triphosphate) on this enzyme in vitro. In addition, tetracycline, an inhibitor of the synthesis of these nucleotides, apparently prevents the change in the quantitative relationship. The relationship between glucose utilization and the hexose phosphates is altered at the transition to Period II of nitrogen starvation. We propose that this change reflects the alteration of the cellular steady state level of an unknown effector of the glucose phosphotransferase system. In contrast to the ATP-hexose phosphate system of shared regulatory effects, the specific effects of the unknown effectors allow the rates of glucose utilization and glycogen synthesis to be altered independently of each other and independently of changes in the rate of glycolysis. This independence allows a greater latitude of response for the individual pathways in more severe metabolic stress or in accommodating the metabolic changes necessary for long term survival.