A mutation in Escherichia coli that mimics diauxie lag.

We have described a mutant of $\text{E.}\text{coli}$ (2S142) which shows a specific inhibition of stable RNA synthesis at 42°. The temperature sensitive lesion differs from the stringent response to amino acid starvation in that the shut off of rRNA synthesis is not associated with an inhibition of protein synthesis. The decay of ppGpp is slow at 42° with little or no pppGpp detectable. This slow decay rate is not observed in the parental strain, D10, or in 2S142 at 30°. Neither 2S142 or D10 are spoT, nor does the temperature sensitive lesion map near the spoT locus. Thus, the effect of the temperature sensitive lesion on ppGpp metabolism and rRNA synthesis seems to resemble a carbon source downshift (diauxie lag) rather than a stringent response to amino acid starvation.     

Accumulation of guanosine tetraphosphate induced by polymixin and gramicidin in Escherichia coli

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA⁺) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.     

The stringent response to unacylated tRNA,energy- and temperature-downshift in Bacillus stearothermophilus

The response of the thermophile Bacillus stearothermophilus to inhibition of tRNA acylation, energy starvation and temperature downshift was characterized. We found that B. stearothermophilus, like other prokaryotic organisms, reacts with the so-called stringent response, which includes the accumulation of the unusual nucleotides guanosine 3′,5′ bis (dipphosphate) [ppGpp] and guanosine 3′-diphosphate, 5′-triphosphate [pppGpp] and concomitantly the reduction of RNA synthesis and growth rate. The amount of (p)ppGpp formed depended on the cause of the stringent response: when tRNA acylation was inhibited (p)ppGpp synthesis was much higher than after energy starvation or temperature downshift whereas RNA synthesis was totally blocked in each case.     

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.     

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     

Inorganic Polyphosphate in Escherichia coli: the Phosphate Regulon and the Stringent Response

Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (P_i), in media deficient in both P_i and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a phoB-containing plasmid, phoB mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during P_i starvation). Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.     

The ObgE/CgtA GTPase influences the stringent response to amino acid starvation in Escherichia coli

The stringent response is important for bacterial survival under stressful conditions, such as amino acid starvation, and is characterized by the accumulation of ppGpp and pppGpp. ObgE (CgtA, YhbZ) is an essential conserved GTPase in Escherichia coli and several observations have implicated the protein in the control of the stringent response. However, consequences of the protein on specific responses to amino acid starvation have not been noted. We show that ObgE binds to ppGpp with biologically relevant affinity in vitro , implicating ppGpp as an in vivo ligand of ObgE. ObgE mutants increase the ratio of pppGpp to ppGpp within the cell during the stringent response. These changes are correlated with a delayed inhibition of DNA replication by the stringent response, delayed resumption of DNA replication after release, as well as a decreased survival after amino acid deprivation. With these data, we place ObgE as an active effector of the response to amino acid starvation in vivo . Our data correlate the pppGpp/ppGpp ratio with DNA replication control under bacterial starvation conditions, suggesting a possible role for the relative balance of these two nucleotides.     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

The effect of alcohols on guanosine 5'-diphosphate-3'-diphosphate metabolism in stringent and relaxed Escherichia coli

The effects of a series of alcohols on the stringent response system of Escherichia coli were studied. The alcohols used could be divided into two groups on the basis of the response of pppGpp and ppGpp to the growth downshift induced by the alcohols. The cells responded to the alcohols, methanol, ethanol, and propanol, as if they were being starved of amino acids. In the stringent strain CP78 these alcohols induced pppGpp and ppGpp accumulation and curtailed RNA synthesis, whereas in the relaxed strain CP79, both of these responses were absent. It was determined that this response was most likely due to an interference by these alcohols with the uptake of amino acids required by these strains. By contrast both stringent and relaxed cells elevated their level of ppGpp and decreased RNA accumulation when treated with butanol or pentanol. This response is similar to the effect of carbon source limitation. It was determined that the elevation of ppGpp in the stringent strain was primarily the result of increased ppGpp synthesis in response to these alcohols. In the relaxed strain the rise in ppGpp was dependent on a decrease in ppGpp degradation coupled with a moderate increase in ppGpp synthesis. This stimulation of ppGpp synthesis in relaxed cells, although small, suggests the existence of an enzyme distinct from stringent factor which is capable of synthesizing ppGpp. Data are presented which suggest that the activity of this enzyme is coupled to the potential for protein synthesis and energy availability of the cell, perhaps being regulated by the overall ratio of unchanged to amino-acylated tRNA.     

Studies on the turnover of endogenous choline-containing phospholipids of cultured neuroblastoma cells

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA+) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.     

The Stringent Response and Cell Cycle Arrest in Escherichia coli

The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.     

Mutations that overcome plasmid-mediated relaxation affect (p)ppGpp

Summary A recombinant plasmid, pMY3, was constructed in this laboratory to express the amber suppressor allele, Su⁺7, of the tRNA^Trp gene from E. coli (Yarus, 1979a). This plasmid also relaxes control of the synthesis of all stable RNA species in its host cell after amino acid deprivation. Guanosine penta and tetra-phosphate (MSII and MSI) concentrations are reduced to about one-half the levels achieved by starving the host cells carrying the cloning vehicle (pMB9) alone.We now show that the relaxation conferred on cells carrying pMY3 can be overcome by at least three different missense mutations at the chromosomal spoT locus. In these stringent, plasmid-carrying strains, the ppGpp levels attained during starvation are equivalent to or higher than that of the host cell carrying the vehicle alone.In vitro mutagenesis of the relaxing plasmid with EMS, followed by transformation and screening for plasmid-bearing stringent cells, yielded four stringent revertants of the relaxing locus. Cells carrying these mutants plasmids all have normal stringent responses to amino acid starvation, and again, elevate (p)ppGpp levels equal to or greater than 80% LS286 (pMB9) levels.Despite pMY3s modest effect on its host's MSI levels during the steady state of starvation, an obvious correlation exists between the concentration of that nucleotide and the host's ability to respond stringently. We therefore believe that the plasmid intervenes in MS metabolism. Measurements of the in vivo rates of decay of MSI and MSII after reversal of isoleucine starvation show that pMY3 has no effect on those reactions. The most likely mechanism of plasmid action is therefore inhibition of MS synthesis.Nonstandard Abbreviations MSI ppGpp - MSII pppGpp - EMS ethyl methane sulfonate - TCA trichloroacetic acid     

Stringent control of protein synthesis in E. coli

The effect of ppGpp on the rate of protein synthesis has been determined in vivo. When the stringent response is triggered and then reversed in isogenic strains carrying either spoT^? or spoT⁺, the mutants lower their elevated ppGpp only slowly, whereas wild type cells do so rapidly. Protein synthesis resumes only after a lag in spoT^? cells but almost immediately in spoT⁺ cells. In spoT^?rel^? double mutants, ppGpp does not accumulate under these conditions and protein synthesis resumes immediately. Inhibition of protein synthesis in spoT^?rel⁺ cells therefore appears to be due to elevation of ppGpp levels and not to any other effect of the spoT^? mutation.     

Purification and properties of stringent factor.

The stringent factor from Escherichia coli is the product of the relA locus. It is the enzyme that catalyzes the synthesis of pppGpp and ppGpp eliciting a pyrophosphate transfer from ATP to the 3'--OH of GTP (or GDP). This protein is responsible for the synthesis of pppGpp and ppGpp in stringent strains in response to an amino acid starvation. In vitro it catalyzes the synthesis of these guanosine compounds in either a ribosome-dependent reaction that requires a particular conformation of the ribosome i.e. the presence of an uncharged tRNA recognizing a codon in the acceptor (A) site of the ribosome or in a ribosome-independent reaction at temperatures under 30 degrees in the presence of only buffer, salts, and substrates. Here we report the purification of the stringent factor to near homogeneity. It is a monomeric protein with a molecular weight of 75,000. The properties of the ribosome-independent reaction are studied and it is shown that the presence of certain acidic proteins, such as the 50 S ribosomal proteins L7 and L12 or casein, or 20% methanol or both stimulates the reaction by creating an environment that together with the low temperature further stabilizes the stringent factor.     

Stringency and relaxation among the halobacteria.

Accumulation of stable RNA and production of guanosine polyphosphates (ppGpp and pppGpp) were studied during amino acid starvation in four species of halobacteria. In two of the four species, stable RNA was under stringent control, whereas one of the remaining two species was relaxed and the other gave an intermediate phenotype. The stringent reaction was reversed by anisomycin, an effect analogous to the chloroamphenicol-induced reversal of stringency in the eubacteria. During the stringent response, neither ppGpp nor pppGpp accumulation took place during starvation. In both growing and starved cells a very low basal level of the two polyphosphates appeared to be present. In the stringent species the intracellular concentration of GTP did not diminish but actually increased during the course of the stringent response. These data demonstrate that (i) wild-type halobacteria can have either the stringent or the relaxed phenotype (all wild-type eubacteria tested have been shown to be stringent); (ii) stringency in the halobacteria is dependent on the deaminoacylation of tRNA, as in the eubacteria; and (iii) in the halobacteria, ppGpp is not an effector of stringent control over stable-RNA synthesis.     

Fatty acid starvation activates RelA by depleting lysine precursor pyruvate

Bacteria undergoing nutrient starvation induce the ubiquitous stringent response, resulting in gross physiological changes that reprograms cell metabolism from fast to slow growth. The stringent response is mediated by the secondary messengers pppGpp and ppGpp collectively referred to as (p)ppGpp or ‘alarmone’. In Escherichia coli, two paralogs, RelA and SpoT, synthesize (p)ppGpp. RelA is activated by amino acid starvation, whereas SpoT, which can also degrade (p)ppGpp, responds to fatty acid (FA), carbon and phosphate starvation. Here, we discover that FA starvation leads to rapid activation of RelA and reveal the underlying mechanism. We show that FA starvation leads to depletion of lysine that, in turn, leads to the accumulation of uncharged tRNA^Lys and activation of RelA. SpoT was also activated by FA starvation but to a lower level and with a delayed kinetics. Next, we discovered that pyruvate, a precursor of lysine, is depleted by FA starvation. We also propose a mechanism that explains how FA starvation leads to pyruvate depletion. Together our results raise the possibility that RelA may be a major player under many starvation conditions previously thought to depend principally on SpoT. Interestingly, FA starvation provoked a ~100‐fold increase in relA dependent ampicillin tolerance.     

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.     

Helicobacter pylori: a Eubacterium Lacking the Stringent Response

Accumulation of 16S rRNA and production of guanosine polyphosphates (pppGpp and ppGpp) were studied during amino acid starvation in three wild-type strains of Helicobacter pylori. All strains exhibit a relaxed phenotype with respect to accumulation of 16S rRNA. This constitutes the first example of a wild-type eubacterium showing a relaxed phenotype. The guanosine polyphosphate levels do not rise as a result of amino acid starvation, as expected for relaxed organisms. However, in both growing and starved cells, basal levels of the two polyphosphates appeared to be present, demonstrating that the enzymatic machinery for guanosine polyphosphate production is present in this organism. These findings are discussed within the framework of the hypothesis that stringent control is a physiological control mechanism more important for the fitness of prokaryotes growing in the general environment than for those that inhabit protected niches.     

Role of a hisU gene in the control of stable RNA synthesis in Salmonella typhimurium

We have previously reported a fivefold reduction in expression of the ilvGEDA operon in a hisU mutant (hisU1820) originally isolated as a histidine regulatory mutant that exhibited derepressed (deattenuated) expression of the his operon. More recently, we have reported that a unitary explanation of the effect of this mutant on amino acid control is complicated by the observation of relaxed control of stable RNA synthesis during carbon/energy source downshifts. In the present study, we report the results of an analysis of the relaxation in control of RNA synthesis in relation to the accumulation of the guanosine polyphosphates, ppGpp and pppGpp. Unexpectedly, we observed that, despite the inability to restrict RNA accumulation upon carbon/energy downshifts, this mutant formed ppGpp at the normal rate. Further, the evidence clearly indicates that the defective control of RNA in this hisU mutant is not owing to an alteration in the spoT gene and that the relA-mediated RNA control is unaltered. However, relaxed RNA synthesis in hisU is suppressed by hyper-elevated levels of ppGpp; thus, an inverse correlation between RNA accumulation and ppGpp level during carbon/energy downshifts is still demonstrable in the hisU mutant. These data led us to the observation that the increased accumulation of stable RNA upon a carbon/energy downshift is apparently the consequence of a hisU-conferred increase in RNA stability.     

Altered specificity of synthesis of guanosine tetraphosphate (ppGpp) and pentaphosphate (ppGpp) by salt-washed ribosomes

Salt-washed ribosomes from $\text{Escherichia coli}$, plus stringent protein, form more ppGpp than pppGpp from GTP at all times, but unwashed ribosomes are shown to synthesize primarily pppGpp as the initial product.