Influence of amino acid starvation on guanosine 5''-diphosphate 3''-diphosphate basal-level synthesis in Escherichia coli.

We observed that the synthesis of basal-level guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA mutants and relA+ relC strains of Escherichia coli decreased in response to amino acid limitation and that this was accompanied by an increase in ribonucleic acid (RNA) synthesis. Addition of the required amino acid to starved cultures of relaxed bacteria resulted in the resumption of ppGpp synthesis and a concomitant decrease in RNA production. Our results indicate that relA mutants retain a stringent factor-independent ribosomal mechanism for basal-level ppGpp synthesis. They also suggest that in relA+ bacteria, stringent factor-mediated ppGpp synthesis and the production of basal-level ppGpp are mutually exclusive. These findings substantiate the hypothesis that there are two functionally discrete mechanisms for ppGpp synthesis in E. coli. Through these studies we have also obtained new evidence which indicates that ppGpp serves as a modulator of RNA synthesis during balanced growth as well as under conditions of nutritional downshift and starvation.     

Eukaryotic ribosomal proteins stimulate Escherichia coli stringent factor to synthesize guanosine 5''-diphosphate, 3''-diphosphate (ppGpp) and guanosine 5''-triphosphate, 3''-diphosphate (ppGpp).

Summary When supplemented with Escherichia coli stringgent factor, 80S ribosomes from various sources failed to support guanosine tetra- and pentaphosphate ((p)ppGpp) synthesis. In contrast, ribosomal proteins from 80S, 60S or 40S particles (mouse embryos, rabbit reticulocytes) crossreacted with the E. coli stringent factor. Significant stimulation of (p)ppGpp synthesis was achieved proteins/ml. These observations may provide additional criteria to detect homologies between eukaryotic and prokaryotic ribosomal proteins.     

Effects of light deprivation on RNA synthesis, accumulation of guanosine 3''(2'')-diphosphate 5''-diphosphate, and protein synthesis in heat-shocked Synechococcus sp. strain PCC 6301, a cyanobacterium.

The rate of total RNA synthesis, the extent of guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) accumulation, and the pattern of protein synthesis were studied in light-deprived and heat-shocked Synechococcus sp. strain PCC 6301 cells. There was an inverse correlation between the rate of total RNA synthesis and the pool of ppGpp, except immediately after a temperature shift up, when a parallel increase in the rate of RNA synthesis and accumulation of ppGpp was observed. The inverse correlation between RNA synthesis and ppGpp accumulation was more pronounced when cells were grown in the dark. Heat shock treatment (47 degrees C) had an unexpected effect on ppGpp accumulation; there was a fairly stable level of ppGpp under heat shock conditions, which coincided with a stable steady-state rate of RNA synthesis even in the dark. We found that the pattern of dark-specific proteins was altered in response to heat shock. The transient synthesis of several dark-specific proteins was abolished by an elevated temperature (47 degrees C) in the dark; moreover, the main heat shock proteins were synthesized even in the dark. This phenomenon might be of aid in the study of cyanobacterial gene expression.     

Mutants of Escherichia coli defective in the degradation of guanosine 5''-triphosphate, 3''-diphosphate (pppGpp).

Summary A new class of mutants of E. coli exhibiting altered metabolism of ppGpp and pppGpp has been isolated, and mapped at a locus designated gpp, near min 83 on the genetic map. These mutants accumulate elevated levels of pppGpp during amino acid starvation or carbon source downshift, and exhibit a reduced rate of pppGpp degradation in vivo. The in vitro evidence suggests that the gpp mutants are defective in a 5-nucleotidase, which specifically hydrolyzes pppGpp to ppGpp. Certain combinations of gpp and spoT mutations are inviable. A gpp spoT double mutant, constructed by employing a leaky spoT mutation, was found to have a slower rate of pppGpp degradation than the gpp mutant alone. This result indicates that spoT also participates in pppGpp degradation. The inviability of certain gpp spoT combinations is attributed to the inability of the double mutants to degrade pppGpp. This is supported by the observation that selection for increased growth rate on the double mutant results in the recovery of relA mutations. Various effects of the gpp mutation upon the pppGpp and ppGpp pools provide additional support for a scheme in which pppGpp is the major precursor of ppGpp.     

Bacteriophage infection interferes with guanosine 3''-diphosphate-5''-diphosphate accumulation induced by energy and nitrogen starvation in the cyanobacterium Anacystis nidulans

Anacystis nidulans accumulates large amounts of guanosine 3'-diphosphate-5'-diphosphate (ppGpp) upon nutritional or energy starvation induced by light-to-dark shift, treatment with carbonylcyanide-m-chlorophenylhydrazone (an uncoupler), or treatment with L-methionine-DL-sulfoximine (an inducer of nitrogen starvation). In contrast to healthy A. nidulans cells, those infected by AS-1 cyanophage do not respond with ppGpp accumulation when starved after about one-third of the complete infection cycle, except, to some extent, under extreme conditions when both nitrogen deprivation and energy deprivation are induced simultaneously (darkening plus L-methionine-DL-sulfoximine treatment). In contrast to cyanophage infection in Anacystis, infection with T4 phage of Escherichia coli CP 78 cells does not affect their accumulation of ppGpp under treatments identical with or similar to those applied in the experiments with Anacystis. This difference in response of phage-infected heterotrophic and photoautotrophic cells to starvation seems to reflect differences in control of nutritional or energy metabolism rather than differences in ability to synthesize ppGpp.     

Iron deprivation in cyanobacteria

Iron is an essential component of electron transport in almost all living organisms. It is particularly important to phototrophs like cyanobacteria because 22–23 irons are required for a complete functional photosynthetic apparatus. Since the low solubility of Fe⁺⁺⁺ above neutral pH in oxic ecosystems severely limits the biological availability of iron to aquatic microorganisms, cyanobacteria and other microbes have developed a number of responses to cope with iron deficiency. Cyanobacterial responses to iron stress include the synthesis of an efficient, siderophore-based system to scavenge iron and the substitution of ferredoxin with flavodoxin. An additional response in cyanobacteria involves the alteration of the light-harvesting apparatus that includes the appearance of a new, iron-stress-induced, photosystem II, chlorophyll-binding protein. Although cytochromec-553 has a potential non-iron-containing replacement in plastocyanin, a copper-containing protein, iron stress appears to favor the utilization of cytochromec-553 because siderophores also bind copper and form a complex that is excluded from the cell.This paper is intended primarily as a review of molecular and physiological responses of actively growing cyanobacterial cultures to conditions of iron stress, where iron is present but essentially insoluble, and to differentiate these responses from iron starvation, where the amount of iron in the system is not sufficient for cell growth.     

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.     

Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate).

A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.     

In vitro synthesis and characterization of guanosine 3',5'-bis(diphosphate)

The intracellular alarmone guanosine 3′,5′-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone’s complex regulatory effects. HPLC analysis and UV detection are used to quantify intracellular ppGpp. The samples analyzed also contain other phosphorylated guanine nucleotides and, therefore, are spiked with a standard ppGpp solution. A rapidly growing number of laboratories are turning to synthesizing the nucleotide in vitro involving time-consuming protocols and yielding only low amounts of ppGpp. The current article provides a protocol for the preparation of large quantities of a ribosome extract that contains high ppGpp synthesis activity. The demonstrated upscaling from shaking flask to bioreactor cultivation involves the continuous and refrigerated harvest of the biomass. ¹³C NMR analysis enabled the structural characterization of the synthesis product and was complemented by mass spectrometry and methods that are commonly used to identify ppGpp.     

Regulation of synthesis of guanosine 3'':5''-cyclic monophosphate in neuroblastoma cells.

The increase in intracellular cyclic GMP concentrations in response to muscarinic-receptor activation in N1E-115 neuroblastoma cells is dependent on extracellular Ca2+ ion. The calcium ionophore A23187 can also evoke an increase in cyclic GMP in the presence of Ca2+ ion. Most (about 85%) of the guanylate cyclase activity of broken-cell preparations is found in the soluble fraction. The soluble enzyme can utilize MnGTP (Km = 55 micrometer), MgGTP (Km = 310 micrometer) and CaGTP (Km greater than 500 micrometer) as substrates. Free GTP is a strong competitive inhibitor (Ki approximately 20 micrometer). The enzyme possesses an allosteric binding site for free metal ions (Ca2+, Mg2+ and Mn2+). The membrane-bound guanylate cyclase is qualitatively similar to the soluble form, but has lower affinity for the metal-GTP substrates. Entry of Ca2+ into cells may increase cyclic GMP concentration by activating guanylate cyclase through an indirect mechanism.     

Role of peptide chain elongation factor G in guanosine 5'-diphosphate 3'-diphosphate synthesis.

In a wild-type strain (relA+) of Escherichia coli, starvation of amino acid led to an immediate cessation of the synthesis of stable ribonucleic acids, together with the accumulation of an unusual nucleotide, guanosine 5'-diphosphate 3'-diphosphate, commonly known as ppGpp. This compound also accumulated during heat shock. When temperature-sensitive protein synthesis elongation factor G (EF-G) was introduced into E. coli NF859, a relA+ strain, the synthesis of ppGpp was reduced to approximately one-half that of wild-type EF-G+ cells at a nonpermissive temperature of 40 degrees C. Furthermore, fusidic acid, an inhibitor of protein synthesis which specifically inactivates EF-G, prevented any accumulation of ppGpp during the heat shock. We suggest that a functional EF-G protein is necessary for ppGpp accumulation under temperature shift conditions, possibly by mediating changes in the function of another protein, the relA gene product. However, EF-G is probably not required for the synthesis of ppGpp during the stringent response, since its inactivation did not prevent ppGpp accumulation during amino acid starvation.     

The synthesis of 2'-methylseleno adenosine and guanosine 5'-triphosphates

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2'-SeCH(3)-uridine and 2'-SeCH(3)-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2'-methylseleno-2'-deoxyadenosine and -guanosine 5'-triphosphates (2'-SeCH(3)-ATP and 2'-SeCH(3)-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.     

The effective synthesis of uridylyl/3''-5''/5-methylcytidylyl/3''-5''/guanosine.

The triester method was adapted to the synthesis of uridylyl/3'-5'/5-methylcytidylyl/3'-5'/guanosine. As the protecting groups 4-methoxy-5,6-dihydro-2H-pyran for 2'-OH and 5'-OH groups of uridine and 2'-OH group of 5-methylcytidine, methoxymethylidene for I:3'-cis-diol system of guanosine, and benzoyl for the amino groups of 5-methylcytidine and guanosine were used. The obtained product was characterised by UV, electrophoresis, chromatography, an enzymatic digestion and alkaline hydrolysis.     

Kinetics of hydrogen-deuterium exchange in guanosine 5'-monophosphate and guanosine 3':5'-monophosphate determined by laser-Raman spectroscopy.

Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in guanosine 5'-monophosphate (5'-rGMP) and guanosine 3':5'-monophosphate (cGMP) were determined as a function of temperature in the range 30-80 degrees C by means of laser-Raman spectroscopy. For each guanine nucleotide the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature: i.e., k psi = Ae-Ea/RT with A = 8.84 X 10(14) h-1 and Ea = 24.6 kcal/mol for 5'-rGMP and A = 3.33 X 10(13) h-1 and Ea = 22.2 kcal/mol for cGMP. Exchange of the 8-CH groups in guanine nucleotides is generally 2-3 times more rapid than in adenine nucleotides [cf. g. j. thomas, Jr., & J. Livramento (1975) Biochemistry 14, 5210-5218]. As in the case of adenine nucleotides, cyclic and 5' nucleotides of guanine exchange at markedly different rates at lower temperatures, with exchange in the cyclic nucleotide being the more facile. Each of the guanine nucleotides was prepared in four different isotopic modifications for Raman spectral analysis. The Raman frequency shifts resulting from the various isotopic substitutions have been tabulated, and assignments have been given for most of the observed vibrational frequencies.     

Correlation between RNA synthesis and basal level guanosine 5'-diphosphate 3'-diphosphate in relaxed mutants of Escherichia coli

Through the use of a new nucleotide extraction procedure, we had previously shown that relaxed mutants of Escherichia coli exhibit a unique response to amino acid starvation (Lagosky, P. A., and Chang, F. N. (1980) J. Bacteriol. 144, 499-508). The basal level amounts of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA and phenotypically relaxed relA+ rplK (relC) strains were shown to decrease at the onset of amino acid limitation and to remain severely depressed throughout the course of the starvation. Upon resupplementation of amino acid-starved relaxed mutants, the production of ppGpp resumes and results in the temporary overaccumulation of this nucleotide beyond its original basal level amount. We now show that the basal level ppGpp content of relaxed bacteria, as well as its subsequent fluctuations in response to amino acid starvation, is inversely correlated with the initial rates of RNA synthesis in these strains. The ability of ppGpp to control the rate of protein synthesis in relA mutants was also examined. It was observed that ppGpp had no apparent direct effect on the initial rates of protein synthesis in relA mutants. The constant inverse correlation which exists between ppGpp content in relA mutants, and their rates of RNa synthesis provide evidence which indicates that basal level ppGpp synthesis has definite physiological significance. It also suggests that the synthesis of basal level ppGpp might be an absolute requirement needed for normal bacterial growth.