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.     

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     

Close linkage of prd and rel genes in Escherichia coli K-12

Summary The prd gene, the mutant allele of which permits growth of E. coli on 1,2-propanediol as sole carbon source, has been located by transduction very close to the rel (RNA control) gene. This close linkage provides a convenient means for the inter-strain transfer of rel.This work was supported in part by the Australian Meat Research Committee.     

Regulation of stable RNA synthesis and ppGpp levels in growing cells of Escherichia coli

Under the balanced condition of growth of E. coli cells, no distinct difference is observed in stable RNA and protein synthesis between CP78 (rel⁺) and CP79 (rel⁻), whereas a considerable difference is present in RNA accumulation between NF161 (rel⁺) and NF162 (rel⁻), where NF161 < NF162. The RNA content of NF161 is lower than that of NF162 in four different cultures with different growth rates. These two sets of isogenic pairs of rel⁺ and rel⁻ strains are commonly used in the study of rel gene function; however, NF161 is a mutant in the spoT gene whose product may be responsible for the degradation of ppGpp. The basal levels of ppGpp in these four strains growing with three different growth rates were examined: NF161 (rel⁺spoT⁻) has a much higher content of ppGpp than do other strains. Furthermore, the contents of ppGpp tend to be lower when the above four strains are growing at a faster rate. Thus a close correlation seems to exist between the content of RNA and the basal level of ppGpp under the condition of balanced growth.     

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     

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.     

Fluorescent monitoring of cellular physiological status depending on the accumulation of ppGpp

For evaluating the physiological status of cells, astringent response network was used. Fluorescence from intact E. coli, which has a plasmid encoding the green fluorescence protein (GFP) under the regulation of rpoS promoter, was monitored. Comparison of the response of different E. coli strains demonstrated an essential role of ppGpp in the expression of GFP, as it activated the rpoS promoter. The physiological status of intact cells, that depends on ppGpp accumulation in response to the nutritional status such as amino acid starvation, could therefore be monitored by measuring fluorescent intensity using this reporter gene.     

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.     

Increased RNA polymerase availability directs resources towards growth at the expense of maintenance

Nutritionally induced changes in RNA polymerase availability have been hypothesized to be an evolutionary primeval mechanism for regulation of gene expression and several contrasting models have been proposed to explain how such ‘passive’ regulation might occur. We demonstrate here that ectopically elevating Escherichia coli RNA polymerase (Eσ⁷⁰) levels causes an increased expression and promoter occupancy of ribosomal genes at the expense of stress‐defense genes and amino acid biosynthetic operons. Phenotypically, cells overproducing Eσ⁷⁰ favours growth and reproduction at the expense of motility and damage protection; a response reminiscent of cells with no or diminished levels of the alarmone guanosine tetraphosphate (ppGpp). Consistently, we show that cells lacking ppGpp displayed markedly elevated levels of free Eσ⁷⁰ compared with wild‐type cells and that the repression of ribosomal RNA expression and reduced growth rate of mutants with constitutively elevated levels of ppGpp can be suppressed by overproducing Eσ⁷⁰. We conclude that ppGpp modulates the levels of free Eσ⁷⁰ and that this is an integral part of the alarmone's means of regulating a trade‐off between growth and maintenance.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Characterization of an E. coli mutant with a thermolabile initiation factor IF3 activity.

Summary A thermosensitive E. coli mutant is described which has at least two defects in vitro: a thermolabile initiation factor IF3 activity and a modified L-phenylalanine: tRNA^Phe ligase (EC 6.1.1.20) activity. These two defects cotransduce and are located near 38 min on the new E. coli map. Thermoresistant revertants showing in vitro reversion for one defect also revert in vitro for the other defect. The thermosensitive mutation is recessive to its wild type allele, and in vitro analysis of wild type/mutant heterodiploïds also show reversion for both defects.     

Differential effect of amino acid starvation on polysome decay in escherichia coli

In arelA⁺ strain ofE. coli starved separately for each of four required amino acids, the intracellular concentration of polysomes decreases as a function of time in all cases: very rapidly in the absence of arginine or leucine, slowly in the absence of threonine or histidine. In a starved isogenicrelA strain, the polysome level is either totally stable or else drops slowly. The decrease in the level, when it occurs, does not significantly affect the polysome size distribution. Models for polysome metabolism in amino acid starved cells are discussed.     

The extraction of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) from Escherichia coli using low pH reagents: a reevaluation.

It has been found that the most widely used method for the extraction of guanosine 5′-diphosphate, 3′-diphosphate (ppGpp) from $\text{E. coli}$ (1 M formic acid at 0°) results in its $\text{in vitro}$ degradation to ppGp and GDP. A comparison with several other extraction procedures indicated that this breakdown is due to the low pH of the reagents used during extraction. This degradation can largely be prevented by using a new extraction technique which involves freezing and thawing of the cells in the presence of lysozyme at a neutral pH followed by treatment with deoxycholate. With this method it is possible to recover from three to five times as much ppGpp from both unstarved and amino acid starved stringent strains of $\text{E. coli}$ as compared with the most widely used formic acid procedure. Consequently, it will be necessary to reevaluate the ppGpp values obtained from cells when formic acid or other low pH reagents were used during extraction.     

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σ^S (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain.     

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.