Nonsense and insertion mutants in the relA gene of E. coli: cloning relA.

We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.     

Functional aspects of bacterial polysomes during limited protein synthesis

The effects of amino acid starvation on the metabolic behavior of polysomes and the size distribution of proteins have been studied in an otherwise isogenic pair of stringent (relA+) and relaxed (relA) strains of Escherichia coli. The stability of polysomes has been analyzed by using two different approaches. First, the process of their degradation has been followed after treating the cells with rifampicin, an inhibitor of the synthesis of all classes of RNA including messenger RNA. Secondly, the process of their assembly has been studied after their previous conversion to monosomes, as induced by glucose deprivation of cells. It is shown that, in either type of bacterial strain, polysomes are continually broken down and re-synthesized during amino acid starvation. However, such polysome turnover is then less rapid than in normally growing bacteria and, moreover, it seems amino acid specific since it occurs at a lower rate during arginine starvation than during histidine starvation, namely, in the relaxed strain. The molecular weight distribution of proteins has been determined after labeling of cells with radioactive methionine and separation of polypeptides by one-dimensional polyacrylamide gel electrophoresis. The average size of polypeptides synthesized in the stringent strain during starvation is quite similar to that measured during normal growth. By contrast, a significant shift towards smaller molecules is observed in the relaxed strain deprived of an essential amino acid. Here again, this reduction of the size of polypeptides seems amino acid specific since it is especially marked during arginine starvation. These results are discussed in terms of ribosomes translocation and premature peptide chain termination in connection with the accuracy of the translational process.     

Involvement of the relA gene product and feedback inhibition in the regulation of DUP-N-acetylmuramyl-peptide synthesis in Escherichia coli.

The regulation of uridine diphosphate-N-acetylmuramyl-peptide (UDP-MurNAc-peptide) synthesis was studied by labeling Escherichia coli strains auxotrophic for lysine and diaminopimelate with [3H]diaminopimelate for 15 min under various conditions. The amounts of [3H]diaminopimelate incorporated into UDP-MurNAc-tripeptide and -pentapeptide by a stringent (rel+) strain were the same in the presence or absence of lysine. Chloramphenicol-treated rel+ cells showed a 2.8-fold increase in labeled UDP-MurNAc-pentapeptide. An isogenic relaxed (relA) strain deprived of lysine showed a 2.7-fold increase in UDP-MurNAc-pentapeptide. Thus, UDP-MurNAc-pentapeptide synthesis is regulated by the relA gene. D-Cycloserine treatment of rel+ and relA strains caused a depletion of intracellular UDP-MurNAc-pentapeptide. Labeled UDP-MurNAc-tripeptide accumulated in D-cycloserine-treated cells of the rel+ and relA strains, suggesting that UDP-MurNAc-pentapeptide is a feedback inhibitor of UDP-MurNAc-peptide synthesis. In lysine-deprived cells, D-cycloserine treatment caused 41- and 71-fold accumulations of UDP-MurNAc-tripeptide in rel+ and relA strains, respectively. A 124-fold increase in UDP-MurNAc-tripeptide occurred in lysine-deprived rel+ cells treated with both chloramphenicol and D-cycloserine. These results indicate that both the relA gene product and feedback inhibition are involved in regulating UDP-MurNAc-peptide synthesis during amino acid deprivation.     

Analysis of the relA gene product of Escherichia coli.

The relA gene product, ATP: GTP 3'-pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome-activated assay at 37 degrees C of 120 mumol guanosine pentaphosphate formed min-1 mg protein-1. The specific activity in a methanol-activated assay at 25 degrees C was found to be 4 mumol guanosine pentaphosphate formed min-1 mg protein-1. These values are about 100 times higher than reported by others. Our further studies of this enzyme led to the following results. Antibodies raised against this enzyme inhibit the ribosome-activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes. The amount of stringent factor in the relA+ strain CP78 is estimated to about 1 copy per 200 ribosomes. The amount of antibody-binding material in CP79 (relA) is at least 5 times lower.     

Cloning and characterization of a relA/spoT homologue from Bacillus subtilis

A PCR-amplified DNA fragment of the relA gene from genomic Bacillus subtilis DNA was used to isolate the entire relA / spoT homologue and two adjacent open reading frames (ORFs) from a λ ZAP Express library. The relA gene, which encodes a protein of 734 amino acid residues (aa), is flanked by an ORF (170 aa) that shares high similarity to adenine phosphoribosyltransferase genes ( apt ), and downstream by an ORF (131 aa) of unknown function. This genetic organization is similar to that in Streptomyces coelicolor A3(2) and Streptococcus equismilis H46A. relA shows significant similarity to the Escherichia coli relA and spoT genes, which are responsible for the synthesis and degradation of the highly phosphorylated guanosine nucleotides (p)ppGpp, triggering the stringent response. Deletion of the relA gene generated a (p)ppGpp⁰ phenotype that demonstrated its essential role in the response to amino acid deprivation and resulted in impaired/lowered induction of proteins involved in stress response as well as amino acid biosynthesis, as judged by two-dimensional gel electrophoresis. The same effects of impaired induction of some σ^B-independent proteins could also be shown in a sigB/relA double mutant, supporting the role of relA in derepression/induction of catabolic and anabolic genes during stringent response.     

Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT.

The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp. strain S14 is subjected to carbon and energy starvation. By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp. strain S14. The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp. S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins. However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation. We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp. strain S14. It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp. S14 spoT mutant strain (csrS1). Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival. On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell. This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied. The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.     

Role of the stringent response in the expression and mechanism of near-ultraviolet induced growth delay

The near-ultraviolet (300-400 nm) induced growth delay of Escherichia coli cells was compared in isogenic relA+ and relA- cells illuminated either in the stationary or the exponential phase. In the latter case: (a) the relA- strains of K12 and B/r exhibited similar maximal growth lags (65 min and 55 min respectively); (b) the maximal lags were 1.5-fold and 4-fold longer, respectively, in the isogenic relA+ strains; (c) the rate of the relA- -dependent guanosine 3',5'-bis(diphosphate) (ppGpp) accumulation was three-times lower in the K12 relA+ strain as compared to the B/r relA- strain: (d) a K12 spoT mutant having an impaired rate of ppGpp degradation had a 2-fold longer lag. On the other hand, when illumination is performed in the stationary phase, isogenic relA+ and relA- cells (B/r or K12) exhibited similar growth lags at any fluences, indicating little if any involvement of the stringent response. These data extend previous observations of T.V. Ramabhadran an J. Jagger [(1976) Proc. Natl Acad. Sci. USA, 73, 59-63] but do not support their conclusion that the stringent response is the main factor responsible for growth delay. By monitoring the intracellular level of ppGpp in relA+ spoT- and relA+ spoT+ growing cells during illumination and the subsequent growth lag we observed that the initial burst of ppGpp decreases slowly all along the lag; in all relA+ strains checked the return of ppGpp to its basal level coincides with the recovery of normal growth. We conclude that it is the accumulation of ppGpp over the basal level due either to the stringent response or to prevention of ppGpp degradation that is responsible for an amplification of the growth lag.     

The relA locus specifies a positive effector in branched-chain amino acid transport regulation.

The regulation of branched-chain amino acid transport and periplasmic binding proteins was studied in Escherichia coli strains which were isogenic except for the relA locus, the gene for the "stringent factor," which is responsible for guanosine tetraphosphate synthesis. The strain containing the relA mutation could not be derepressed for the synthesis of leucine transport or binding proteins when shifted from a medium containing all 20 amino acids in excess to one in which leucine was limiting. The relA+ strain showed normal derepression under these conditions.     

Temperature-sensitive growth and decreased thermotolerance associated with relA mutations in Escherichia coli

The relA gene of Escherichia coli encodes guanosine 3',5'-bispyrophosphate (ppGpp) synthetase I, a ribosome-associated enzyme that is activated during amino acid starvation. The stringent response is thought to be mediated by ppGpp. Mutations in relA are known to result in pleiotropic phenotypes. We now report that three different relA mutant alleles, relA1, relA2, and relA251::kan, conferred temperature-sensitive phenotypes, as demonstrated by reduced plating efficiencies on nutrient agar (Difco) or on Davis minimal agar (Difco) at temperatures above 41 degrees C. The relA-mediated temperature sensitivity was osmoremedial and could be completely suppressed, for example, by the addition of NaCl to the medium at a concentration of 0.3 M. The temperature sensitivities of the relA mutants were associated with decreased thermotolerance; e.g., relA mutants lost viability at 42 degrees C, a temperature that is normally nonlethal. The spoT gene encodes a bifunctional enzyme possessing ppGpp synthetase and ppGpp pyrophosphohydrolase activities. The introduction of the spoT207::cat allele into a strain bearing the relA251::kan mutation completely abolished ppGpp synthesis. This ppGpp null mutant was even more temperature sensitive than the strain carrying the relA251::kan mutation alone. The relA-mediated thermosensitivity was suppressed by certain mutant alleles of rpoB (encoding the beta subunit of RNA polymerase) and spoT that have been previously reported to suppress other phenotypic characteristics conferred by relA mutations. Collectively, these results suggest that ppGpp may be required in some way for the expression of genes involved in thermotolerance.     

Stringent regulation of the synthesis of a transfer ribonucleic acid biosynthetic enzyme: transfer ribonucleic acid(m5U)methyltransferase from Escherichia coli.

This paper describes the regulation of a transfer ribonucleic acid (tRNA) biosynthetic enzyme, the tRNA(m5U)methyltransferase (EC 2.1.1.35). This enzyme catalyzes the formation of 5-methyluridine (m5U, ribothymidine) in all tRNA chains of Escherichia coli. Partial deprivation of charged tRNAVal can be imposed by shifting strains carrying a temperature-sensitive valyl-tRNA ligase from a permissive to a semipermissive temperature. By using two such strains differing only in the allelic state of the relA gene, it was possible to show the tRNA(m5U)methyltransferase to be stringently regulated. Upon partial deprivation of charged tRNAVal, the differential rate of tRNA(m5U)methyltransferase synthesis was found to decrease in a strain with stringent RNA control (relA+), whereas it increased in the strain carrying the relA allele. This increase of accumulation of tRNA(m5U)methyltransferase activity required protein synthesis. Thus, when tRNA is partially uncharged in the cell, the relA gene product influences the expression of tRNA(m5U)methyltransferase gene.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

Purification of dihydrofolate reductase amplified in Escherichia coli K-12

The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al. (1983) J. Biochem. 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid. Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography. By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site. Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.     

The role of the omega subunit of RNA polymerase in expression of the relA gene in Escherichia coli

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level.     

A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2)

In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.