Movement of ribosomes over messenger RNA in polysomes of rel + and rel - Escherichia coli strains

The in vitro movement of ribosomes over messenger RNA was studied in both the presence and the absence of protein synthesis. For this purpose, labeled polysomes were extracted from rel⁺ and rel^? strains of Escherichia coli grown in the presence of radioactive uracil and incubated in a cell-free system containing tRNA, amino acids, soluble enzymes and a source of energy. The gradual conversion of the labeled polysomes into monosomes and ribosomal subunits was followed by subjecting the reaction mixture to sucrose gradient sedimentation after various incubation times and measuring the radioactivity present in the three relevant ribosomal fractions.It was found that when the conditions of incubation allow protein synthesis to occur, polysomes extracted from rel⁺ and rel^? cells are converted mainly into free monosomes, which can be made to dissociate into subunits by high-sodium or low-magnesium ion concentrations. Under conditions in which protein synthesis cannot occur because a mutant aminoacyl-tRNA synthetase has been rendered inactive, polysome conversion still occurs, though to a reduced extent. When the products of such residual run-off are examined, however, a difference is manifest between polysomes extracted from rel⁺ and from rel^? strains: whereas the polysomes from the rel^? strain are still converted into free monosomes even in the absence of protein synthesis, the polysomes from the rel⁺ strain are now converted mainly into subunits. It can be inferred, therefore, that ribosomes from rel^? bacteria, but not those from rel⁺ bacteria, continue movement over messenger RNA in the absence of protein synthesis.Studies of mixed extracts from rel^? and rel⁺ bacteria have shown that the character of the run-off process does not depend on the source of tRNA and soluble enzymes; the proportions of monosomes and subunits among the run-off products formed in the absence of protein synthesis depend only on the source of the polysomes. It is suggested that the mutation of the rel gene alters the functional architecture of ribosomes.     

Specificity of the amino acid transfer reaction inE. coli strains carrying different alleles of the RNA control (RC) gene

Summary The idea has been tested here that the aberration in amino acid controlled regulation of RNA synthesis in a mutant strain ofE. coli might reflect a major breakdown in the specificity of transfer of amino acids to S-RNA. For this purpose, S-RNA and amino acid activating enzymes were extracted from bacteria carrying either the normalRC ^st or the aberrantRC ^rel allele of the RNA control gene. The purified S-RNA preparations were first charged enzymatically with one or more of the 20 standard amino acids, then oxidized with periodate, and finally reisolated and retested for their residual capacity to accept an amino acid that was absent from the preliminary charging mixture. If preliminary charging transferred an amino acid to a non-cognate S-RNA species belonging to an absent amino acid, then the acceptor capacity for the missing amino acid would survive periodate oxidation and reveal its presence on recharging with that amino acid after post-periodate reisolation of the S-RNA. The results presented here show that there does not appear to exist any such major breakdown of transfer specificity in eitherRC ^st orRC ^rel bacteria: preliminary charging of the S-RNA fromRC ^rel bacteria with 19 of the 20 standard amino acids by use of the homologous amino acid activating enzymes does not afford protection against periodate oxidation for any appreciable fraction of the acceptor capacity for the absent 20th amino acid (when that amino acid is either methionine or arginine). It is unlikely, therefore, that thecatholic inducer, postulated to explain the continued RNA synthesis ofRC ^rel amino acid auxotrophs in the absence of their growth requirement, is one of the 20 standard amino acids.This investigation was supported by Public Health Service Research Grant CA 02129, from the National Cancer Institute.     

The relationship between guanosine tetraphosphate,polysomes and RNA synthesis in amino acid starved escherichia coli

ArelA⁺ strain ofE. coli with four amino acid requirements was starved separately for each amino acid, after which the levels of polysomes, guanosine-5-diphosphate-3-diphosphate and the residual net synthesis of RNA were determined. The polysome level and guanosine-5-diphosphate-3-diphosphate production were coordinately affected by starvation for the different amino acids, whereas no correlation was found between these two parameters and residual RNA synthesis. The main conclusion stemming from these results is that guanosine-5-diphosphate-3-diphosphate cannot act as the sole effector molecule in stringent control of RNA synthesis.     

The relA locus and the regulation of lysine biosynthesis in Escherichia coli

Summary The allelic state of relA influences the phenotype of Escherichia coli strains carrying the lysA22 mutation: lysA22 relA strains are Lys^– where lysA22 relA ⁺ strains grow (slowly) in the absence of lysine. This physiological effect has been related to an effect of the expression of the relA locus on the regulation of lysine biosynthesis. The fully derepressed levels of some lysine enzymes (aspartokinase III, aspartic semialdehyde dehydrogenase, dihydrodipicolinate reductase) are observed under lysine limitation only in rel ⁺ strains. And the induction of DAP-decarboxylase by DAP is much higher in rel ⁺ than in rel ^– strains when an amino acid limitation of growth is also realised. These results are in agreement with the hypothesis of Stephens et al. (1975) on a possible role of the stringent regulation as a general signal for amino acid deficiency.     

The regulation of the ammonia assimilatory enzymes in Rel+ and Rel- strains of Salmonella typhimurium

Summary The influence of the relA1 mutation on the regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 1.4.1.3), was examined. When cells grown in rich media (either Luria broth or glucose-ammonia plus casamino acids) were transferred to a glucose-ammonia medium, the relA mutant failed to resume growth and did not have the same increase in any of the assimilatory enzyme activities as the rel ⁺ strain. This effect was particularly dramatic for glutamate dehydrogenase, which increased 6-fold in the rel ⁺ strain. Measurements of the guanosine nucleotide concentrations showed that the rel ⁺ strain had a ppGpp concentration about 9 times that of the relA mutant 5 min after the shift to minimal medium. These results are consistent with those for other biosynthetic enzymes and show that the ammonia assimilatory enzymes require a relA product for their synthesis during shifts from rich to minimal media. In addition, we examined the response of these strains to a change in nitrogen source. The relA mutant again failed to resume growth after a shift from glucose-ammonia to glucose-arginine medium. Even though the ppGpp concentration did not increase, the rel ⁺ strain grew and increased glutamine synthetase activities about 2-fold. These changes in the absence of increased ppGpp levels suggest that some other relA-mediated function is important during this change in nitrogen source.     

Relaxed mutants ofSerratia marcescens SM-6. Biochemical traits and relevance of therel + allele for the formation of exoenzymes

Serratia marcescens SM-6 when starved for a required amino acid stops synthesizing protein and RNA and accumulates two nucleotides which cochromatograph with ppGpp and pppGpp. These features are characteristic of bacterial strains with stringent RNA control (rel ⁺). Two independent mutants were isolated which resemble relaxed (relA) mutants ofEscherichia coli; they continue to synthesize RNA and accumulate neither ppGpp nor pppGpp when deprived of the required amino acid. The extracellular enzyme activities (nuclease, protease, lipase) of the relaxed mutants are about the same as those of the parental stringent strain when studied under standard growth conditions. Exoenzyme-deficient (nuc; prt) and exoenzyme-hyperproducing (nuc ^su) mutants were isolated from both stringent and relaxed strains ofS. marcences SM-6 and no change of the cellular ability to form ppGpp and pppGpp could be observed. From these results it appears that the formation of exoenzymes ofS. marcescens SM-6 is independent of stringent/relaxed RNA control.Abbreviations cpd cyclic nucleotide phosphodiesterase deficient - nuc nuclease deficient - nuc ^su nuclease hyperproducing - prt protease deficient - rel relaxed control - spo ppGpp deficient (spot less) - ppGpp guanosine tetraphosphate - pppGpp guanosine pentaphosphate - TCA trichloroacetic acid - OD optical density - EU enzyme units     

Cell volume change of Escherichia coli under stringent control apparent increase of K in rel cells

With several pairs of rel⁺ and rel⁻ strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K⁺ and the rate of uptake of ⁴²K⁺ were investigated. In the early phase of the experiments, the intracellular concentration of K⁺ was estimated by the conventional method in which the cell volume per A₆₆₀ value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K⁺ concentration of rel⁺ cells was kept almost constant, while that of rel⁻ cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A₆₆₀ changed only slightly in CP78 (rel⁺) cells, while it increased markedly in CP79 (rel⁻) cells after the exposure to amino acid starvation. Reestimation of the K⁺ concentrations based on the estimated respective values of cell volumes per A₆₆₀ revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel⁺ cells was arrested upon amino acid starvation whereas that in the rel⁻ cells was not. The ⁴²K⁺ uptake by the rel⁺ cells was depressed upon amino acid starvation, whereas that by the rel⁻ cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K⁺ concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A₆₆₀ of the culture, was pointed out.     

Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC)

Cell growth limitation is known to be an important condition that enhances l-valine synthesis in Corynebacterium glutamicum recombinant strains with l-isoleucine auxotrophy. To identify whether it is the limited availability of l-isoleucine itself or the l-isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of l-valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel ⁻ ) C. glutamicum ΔilvA ΔpanB Δrel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and l-valine synthesis in rel ⁺ and rel ⁻ strains under conditions of l-isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in l-valine synthesis were observed in both rel ⁺ and rel ⁻ cells exposed to l-isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and l-valine synthesis in cells during the stationary growth phase.     

Nonsense suppression in aminoacyl-t-RNA limited cells

Summary A number of nonsense alleles of lacZ exhibit phenotypic suppression (as much as a sixteen-fold increase in leakiness) during partial limitation for certain aminoacyl-tRNA species in relA mutant cells. Each responsive allele has its individual pattern of response to limitation for one or more amino acids or aminoacyl-tRNA's. The phenotypic suppression occurs only during limitation, and ceases once limitation is reversed. Suppression is much reduced by the presence of the relA⁺ allele or an allele of rpsL which restricts ribosomal ambiguity. In one case, the suppressed product has been identified by radioimmune assay and gel electrophoresis, and is a full-length lacZ protomer. Mechanisms are discussed whereby aberrations of translation at codons calling for an aminoacyl-tRNA species in short supply might lead to readthrough of a nearby nonsense codon.     

Role of relA mutation in the survival of amino acid-starved Escherichia coli

Amino acid-starved cells of Escherichia coli relA ⁺, which contain a large number of glycogen particles, are able to survive in phosphate buffer for a longer time period than their relaxed counterparts. With regard to NH ₄ ⁺ starvation differences in the survival of both strains were not found. NH ₄ ⁺ starved cells of E. coli relA are able to synthesize glycogen but amino acid-starved cells of the relA strain are not. We suggest that the synthesis of glycogen triggered by guanosine tetraphosphate during amino acid starvation is responsible for the prolonged viability of the E. coli relA ⁺ strain.Abbreviations ppGpp guanosine tetraphosphate     

Cyclic AMP can replace the relA-dependent requirement for derepression of acetohydroxy acid synthase in E. coli K-12.

Derepression of the isoleucine and valine biosynthetic enzymes was strongly impaired in a relA strain of E. coli K-12 grown in an amino acid-glucose medium. The expression of the isoleucine and valine operons during leucine starvation was markedly defective in the relA mutant as compared to an isogenic rel⁺ strain. Downshift to a poor carbon and energy source or the addition of cyclic AMP to the glucose medium allowed normal derepression in the relA mutant of one of the isoleucine and valine enzymes, acetohydroxy acid synthase. The other isoleucine and valine enzymes failed to derepress under these conditions, in contrast to the high enzyme levels in the rel⁺ parent. No increase in acetohydroxy acid synthase was found in relA cya or relA crp strains during glycerol or pyruvate downshift. Cyclic AMP allowed derepression in the relA cya mutant but not in the relA crp strain. These data strongly suggest that the relA requirement for normal expression of acetohydroxy acid synthase can be replaced by cyclic AMP.     

The relA/spoT-Homologous Gene in Streptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.     

Addition of nutrients induce a fast loss of flocculation in starved cells of Saccharomyces cerevisiae

Incubation of flocculent cells of a brewing strain of Saccharomyces cerevisiae of the NewFlo phenotype for 8–12 h, in the absence of either carbon or nitrogen source, did not induce a loss of flocculation, although an increase (about two times) in the number of cells occurred in nitrogen starved cells. The addition of glucose or ammonium sulphate to carbon or nitrogen starved cells, respectively, triggered a rapid loss of flocculation.     

CHEMOSENSORY RESPONSES OF THE SYMBIOTIC DINOFLAGELLATE SYMBIODINIUM MICROADRIATICUM (DINOPHYCEAE)1

Motile Symbiodinium microadriaticum (Freudenthal 1962) were attracted to a variety of nitrogen-containing compounds, including ammonium, nitrate, urea and some amino acids. No chemosensory response to phosphate, sulphate, vitamins, trace metals or sugars was evident. Motile algae responded to concentrations of ammonium, nitrate, and urea at least as low as 10^?6 M. High concentrations (≥ 10^?2 M) of ammonium appeared to inhibit attraction of motile algae. Calculations using ammonium release rates from various aposymbiotic hosts suggest that motile S. microadriaticum can respond to released ammonium ca. 1 cm from the source. Cultured algae were not attracted to combined nitrogen cues for at least 2 days after inoculation into seawater with dissolved combined low nitrogen. Algae freshly isolated from starved animals were normally motile the day following isolation and attracted to ammonium and nitrate when maintained in seawater containing < 1 μM ammonium and nitrate. The algae lost their ability to orient to nitrogen attractants the day after incubation into culture medium containing high levels of ammonium and nitrate. These results suggest that chemosensory behavior is suppressed when nutrients are present in the ambient medium or are stored by the alga. There were few differences in chemosensory abilities in different strains of S. microadriaticum to the attractants assayed, suggesting that selection for a particular strain by a host species may not be due to differential chemosensory ability or cues. However, the absence of chemical attraction of motile S. microadriaticum to infected hosts may act to preserve strain selection occurring at other steps in the infection process of aposymbiotic hosts.     

Endogenous metabolism ofStaphylococcus aureus

Some of the reactions of endogenous metabolism ofStaphylococcus aureus were examined and the level of endogenous substrate was found to be related to oxygen consumption. In starved cells, the amount of free amino acids, protein and RNA decreased while that of DNA increased. The cells consumed mainly glutamic and aspartic acids and, to a lesser degree, alanine, glycine and serine, while leucine/isoleucine, methionine, tyrosine and phenylalanine were released into the medium. The degradation of RNA by starved cells was suppressed by Mg²⁺. Resting cells ofStaphylococcus aureus oxidized adenosine, guanosine, uridine, adenine, guanine and ribose. After 3 h of starvation at 37°C the viability of cells was not affected although 40.6% amino acids, 8.5% protein and 36.5% RNA had been consumed. Presented in part at the XVIIth Meeting of the Polish Society for Microbiology, Warsaw, September 1970.     

Thermolability of the stringent factor in rel mutants of Escherichia coli

The stringent factor extracted from several independently isolated rel^- mutants is more thermolabile than the stringent factor extracted from the parental rel⁺ strain. This thermolability is characteristically different in each of the mutants. This strongly suggests that the stringent factor is the product of the rel gene.     

Isolation and characterization of prototrophic relaxed mutants of Klebsiella pneumoniae

Summary A new selection procedure has been developed for isolating prototrophic relaxed mutants of Klebsiella pneumoniae. Two mutants were isolated. One of them showed a fully relaxed phenotype, while the other one behaved in a semi-relaxed way.The wild-type strain, as well as the rel mutants exerted similar patterns to their E. coli counterparts in RNA, protein, ppGpp and pppGpp accumulation during amino starvation, carbon source shift-down and nitrogen starvation. Both mutants became stringent after introducing an F-factor carrying the relA ⁺ allele from Escherichia coli. The relaxed phenotype could be recovered by curing the F-factor. Some of the pleiotropic consequences of rel mutations found in E. coli are present in the Klebsiella mutants also while some of them are absent.The mutants are defective in dinitrogen fixation after the exhaustion of limiting ammonium from the culture medium. However, their merodiploid derivatives, carrying the E. coli relA ^- allele, showed the wild-type level of nitrogenase activity under the same conditions.Fellow of the 6th International Training Course jointly sponsored by UNDP/UNESCO Hungarian Academy of Sciences. Present address: Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und Experimentelle Therapie Jena, Beuthenberg Str. 11, DDR-69 Jena     

Reduction of Methionine Sulfoxide by Plants

A relaxed (rel) mutant was found among thirty spontaneous thiopeptin-resistant isolates of Streptomyces antibioticus strain 3720, an actinomycin-producing strain, which showed severely reduced ability to accumulate ppGpp during a nutritional shift-down. The pool size of GTP decreased markedly in the parental strain, but to a lesser extent in the rel mutant. The rel mutant did not show the induction of an enzyme, phenoxazinone synthase, which is involved in the biosynthesis of actinomycin. No negative effect of the rel mutation was observed on a constitutive enzyme, kynurenine formamidase, which also plays a role in actinomycin synthesis. The mutant also failed to produce melanin, but still retained the ability to form aerial mycelium and spores, although the onset of the formation of aerial mycelium was markedly delayed. Neither the phenoxazinone synthase activity nor the kynurenine formamidase activity was affected by ppGpp in vitro. It is suggested tha the stringent response (ppGpp) may be generally essential for the induction of enzymes involved in secondary metabolism.