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     

Variations in ppGpp levels in Rhodopseudomonas spheroides during adaptation to decreased light intensity

The guanosine nucleotides ppGpp and pppGpp have been found in the photosynthetic bacterium $\text{Rhodopseudomonas spheroides}$ supporting a suggestion that these are ubiquitous compounds in bacteria. ppGpp levels undergo an abrupt rise coincident with the cessation of RNA accumulation which occurs in this bacterium following a down-shift in incident light intensity. An abrupt decrease in the rate of [³H] uridine uptake into acid-precipitable material characteristic of light shift-down is also coincident with the rise in ppGpp levels. A regulatory role for this nucleotide in $\text{R. spheroides}$ similar to that proposed in other bacteria is suggested.     

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.     

Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. II. Fraction of RNA polymerase engaged in the synthesis of stable RNA at different steady-state growth rates

The relative rates of stable RNA synthesis (rate of stable synthesis/rate of total RNA synthesis) were determined for Escherichia coli$\text{B}\text{r}$ growing in succinate (μ = 0.69 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) media. The relative rates were 0.29, 0.50 and 0.66 at these growth rates. From the relative rates, the fraction of RNA polymerase engaged in the synthesis of stable RNA, ψ_s, was calculated to be 0.22, 0.36 and 0.48, respectively, by taking into account the difference between the RNA chain growth rate of stable and that of unstable RNA. The relationship between these ψ_s values and μ and our previously determined chain growth rate of stable RNA has two implications for the control of RNA synthesis during a nutritional shift-up: (1) the increase in the net rate of RNA synthesis after a shift-up results from a transfer of RNA polymerase molecules from unstable to stable RNA genes, and a concomitant increase in the stable RNA chain growth rate, but does not require an activation of RNA polymerase; (2) the synthesis of functioning RNA polymerase enzymes is subject to a growth rate-dependent control.     

Novel nucleotides from E.coli isolated and partially characterized

Two new nucleotides have been found in the formic acid extracts of $\text{Escherichia}\text{coli}$, $\text{Clostridium}\text{botulinum}$, $\text{Bacillus}\text{subtilis}$ and $\text{Rhodospirillum}\text{rubrum}$ isolated during log phase growth. In $\text{E.}\text{coli}$ the compounds are present at all times during cell growth but increase in amount during interruption of aeration and transition to stationary phase. They migrate close to ppGpp during one dimensional chromatography on PEI cellulose but are clearly separated from ppGpp by paper chromatography. The compounds are unstable on PEI cellulose and purification was effected by chromatography on A25 Sephadex ion exchange columns. Preliminary characterization indicates that the predominant compound is a dinucleoside polyphosphate and that both compounds contain a modified adenosine nucleoside.     

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Regulation of ribonucleic acid synthesis by `magic spot'' nucleotides

During the inhibition of RC(str), but not RC(rel) mutants of Escherichia coli by trimethoprim the unusual nucleotides MSI (guanosine tetraphosphate, ppGpp) and MSII rapidly accumulated. The production of these nucleotides was not dependent on the addition of nucleotide base supplements to RC(str) cultures before trimethoprim, and the MSI nucleotide concentrations in non-supplemented or purine-supplemented cultures were comparable with the concentrations obtained when the cells were inhibited with l-valine (1g/l). Rifampicin rapidly decreased MSI and MSII nucleotide concentrations in trimethoprim-inhibited cultures to the basal values. Several situations were noted, in which MS nucleotide concentrations in trimethoprim-inhibited RC(str) cells could be drastically lowered without giving rise to an immediate resumption of stable RNA accumulation. If RC(str) mutants were first inhibited with trimethoprim and then given purines 15min later, MS nucleotide concentrations fell rapidly, because of a temporarily enhanced rate of accumulation of stable RNA. However, after a further 5min, RNA accumulation stopped, though MS nucleotide concentrations remained low. Also, if either glycine or methionine were added to trimethoprim-inhibited cultures supplemented with purines, RNA accumulation did not resume, though MS nucleotide concentrations rapidly declined. With both amino acids present, there was both a decline in MS nucleotide concentration and a resumption in stable RNA synthesis. These findings suggest that MSI nucleotide concentrations in trimethoprim-inhibited bacteria are not the sole factors in the control of stable RNA synthesis. It is possible that, during the period when the RC(str) cells contained high concentrations of MS nucleotides, some factor important in the MSI-mediated control of stable RNA synthesis was irreversibly inactivated. However, as antibiotics (e.g. chloramphenicol) both abolished high MS nucleotide concentrations and permitted a rapid resumption of stable RNA accumulation in the same conditions, it is more likely that an additional control mechanism has come into play.     

Regulation of the in vitro synthesis of the alpha-peptide of beta-galactosidase directed by a restriction fragment of the lactose operon.

The regulation of the $\text{in vitro}$ synthesis of the N-terminal portion of the β-galactosidase molecule (α-peptide) has been investigated using DNA fragments of the lactose operon as template. DNA fragments of about 789 base pairs were isolated after endonuclease (Hin II) digestion of either λplac5, λh80dlacp^s or λh80dlacUV5 phage DNA or DNA from the recombinant plasmid PMC3. The regulation of the expression of these fragments is similar to that observed for the synthesis of β-galactosidase using total phage or plasmid DNA as template, indicating that the regulatory regions on the fragments are intact and functional. Thus, the synthesis of the α-peptide required an inducer due to the presence of $\text{lac}$ repressor in the $\text{E. coli}$ S-30 extract used. In addition a dependency on adenosine 3′,5′-cyclic monophosphate (cAMP)¹ for α-peptide synthesis was obtained with the fragments isolated from λplac5 and λh80dlacp^s DNAs, whereas little effect of cAMP was seen with the fragment isolated from λh80dlacUV5 phage DNA or PMC3 plasmid DNA containing a UV5 promotor region. However, a significant difference in the effect of guanosine-3′-diphosphate-5′-diphosphate (ppGpp) was observed. With the total phage DNA as template, ppGpp resulted in a 2–4 fold stimulation whereas with the fragment, or PMC3 plasmid DNA, directed synthesis of the α-peptide no significant stimulation by ppGpp was seen.     

Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. 1. Ribosomal RNA chain growth rates

The chain growth rate for ribosomal RNA was determined for Escherichia coli$\text{B}\text{r}$ growing in succinate (μ = 0.69 doublings/h), glucose (μ = 1.36) and glucose/ amino acids (μ = 2.10) medium. With increasing bacterial growth rate the chain growth rate increases from 4400 to 6300 nucleotides/min. These values are almost twofold higher than the chain growth rate reported for messenger RNA; this implies that, following a nutritional shift-up, the transfer of a relatively small number of RNA polymerase molecules from unstable to stable RNA genes along with the increase in the stable RNA chain growth rate is sufficient to account for the abrupt increase in the net rate of RNA synthesis. Furthermore, our calculations indicate that the linear density of polymerase molecules on the ribosomal DNA template increases with the bacterial growth rate, such that in rapidly growing bacteria all ribosomal RNA genes (48 copies at μ = 3) are nearly saturated with RNA polymerase.     

The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the beta beta' subunits of RNA polymerase following a nutritional shiftup of Escherichia coli.

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the beta and beta' subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT-). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the beta and beta' subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT- strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and beta and beta' subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the beta and beta' subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT- strain versus the timing when the new rates of stable RNA, ribosomal protein, and beta and beta' subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.     

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.     

Increased levels of rat hepatic nuclear free and engaged RNA polymerase activities during liver regeneration.

Rat liver nuclei, seventeen hours after partial hepatectomy, showed a two to three-fold increase in total RNA synthesis $\text{in vitro}$ over the sham operated controls. When tested with exogenous synthetic template, this was found to be mainly a reflection of increased levels of both the nuclear free and engaged RNA polymerase activities $\text{per se}$. It was also observed that there was a greater stimulation of the species of RNA polymerase that are α-amanitin resistant than sensitive (3.2 μg/ml). This observation was further confirmed by DEAE-Sephadex column chromatography of the solubilized nuclear free and engaged RNA polymerases and found RNA polymerase I and IIIa were the major species greatly stimulated during this period of liver regeneration. These data suggest not only that there exists a sensitive equilibrium between the nuclear free and engaged RNA polymerases; they also suggest the possibility that RNA polymerase itself may play a positive role in the regulation of gene expression.     

Effect of unusual guanosine nucleotides on the activities of some Escherichia coli cellular enzymes

Unusual guanosine nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp, also known as MSI) and guanosine 5'-diphosphate 3'-monophosphate (ppGp, also known as MSIII) accumulate to high concentrations in wild-type cells of Escherichia coli during amino acid starvation. We reported here that both nucleotides strongly inhibit the activity of enzymes IMP dehydrogenase and adenylosuccinate synthetase, the first enzymes of the guanylate and adenylate biosynthetic pathways. In both cases, ppGP (MSII) is a stronger inhibitor than ppGpp (MSI). On the other hand, these two nucleotides exhibited opposite effects on the activity of phosphoenolpyruvate carboxylase, the enzyme that utilizes phosphoenolpyruvate. At their respective physiological concentrations, the activity of phosphoenolpyruvate carboxylase is activated by ppGpp and inhibited by ppGp.     

Phosphorylated guanosine derivatives of eukaryotes: Regulation of DNA-dependent RNA polymerases I,II, and III in fungal development

Three phosphorylated guanosine derivatives designated HS-1, HS-2 and HS-3 synthesised during active protein synthesis in the water-mould, $\text{Achlya}$ sp (1969) were shown to regulate the enzymatic activities of nucleoplasmic and nucleolar DNA-dependent RNA polymerases (RNAP-I, II and III) from both $\text{Achlya}$ and another unrelated water-mould, $\text{Blastocladiella emersonii}$. These HS compounds were without effect on $\text{E. coli}$ DNA-dependent RNA polymerase holoenzyme. The most potent of the three compounds was HS-3 which inhibited the activity of all enzymes completely at 100 μg/ml. HS-1, on the other hand, activated maximally at 1 to 10 μg/ml. HS-1 activation (3-fold) was restricted to enzyme III, and it had only partial inhibitory effects on enzymes I and II. The pattern of synthesis of HS-compounds throughout the 20-hour asexual growth cycle of the organism correlated with the detectable levels of the different RNA polymerases of $\text{Achlya}$.     

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.     

Inefficiency of the stringent response in the fungus Mucor

Removal of a required amino acid from the growth medium or addition of cycloheximide caused an immediate stoppage of growth and protein synthesis in the fungus $\text{Mucor}\text{racemosus}$. However, RNA synthesis persisted for several hours at rates that only gradually decreased under the same circumstances. An analysis of the major classes of RNA synthesized during the first hour of treatment showed that cycloheximide preferentially inhibited rRNA synthesis, whereas amino acid starvation slowed synthesis of all RNA species uniformly. Neither treatment affected the percentage of mRNA synthesized. The partial and delayed effects of amino acid starvation and cycloheximide treatment on RNA synthesis reported here suggest the absence of or the gross inefficiency of a classical stringent response in $\text{M.}\text{racemosus}$.     

Altered metabolism of ppGpp in quinone-treated E. coli: Possible role of aminoacyl-tRNA synthetases

The bacteriostatic quinone 6-amino-7-chloro-5,8-dioxoquinoline inhibits leucyl-tRNA synthetase in vivo and in vitro (Ogilvie et al. Biochim. Biophys. Acta $\text{407}$, 357–364; 1975). In this report it is shown that the quinone also interferes with the metabolism of ppGpp. Quinone treatment of E. coli MRE 600 causes the same phenotypic pattern as found in spoT^? mutants: overproduction of ppGpp and a drastic increase of its half-life; the formation of pppGpp, the possible degradation product of ppGpp, is blocked. A model is discussed to explain how the inhibition of leucyl-tRNA synthetase could account for the altered metabolism of ppGpp.     

Isopycnic analysis of intact cells — I: Escherichiacoli over its growth curve

Two computer models for the decline of ribosomal RNA in late exponential phase $\text{E.}\text{coli}$ are tested isopycnographically. The model in which the excess r-RNA is scavenged to support the last stages of cell division is discarded, whereas the experimental data support the model in which r-RNA production is halted and the r-RNA is diluted among the cells as they continue their final division. This plus the pycnotic profile of $\text{E.}\text{coli}$ relA^? support previous work pertaining to control of the genome by guanosine-3′-diphosphate-5′-diphosphate (ppGpp). Other evidence suggests the possibility that part of the genome is also under separate control.     

Translational control of the expression of the β subunit gene of E., coli RNA polymerase

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the $\text{in}\text{,}\text{vitro}$ synthesis of fMet-Val, the N-terminal dipeptide of the β subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of β,β′ synthesis is at the level of translation. L factor ($\text{nusA}$ gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.