ppGpp: stringent response and survival

Adaptation to any undesirable change in the environment dictates the survivability of many microorganisms, with such changes generating a quick and suitable response, which guides the physiology of bacteria. During nutritional deprivation, bacteria show a stringent response, as characterized by the accumulation of (p)ppGpp, resulting in the repression of stable RNA species, such as rRNA and tRNA, with a concomitant change in colony morphology. However, genes involved in amino acid biosynthesis become over-expressed to help bacteria survive under such conditions. The survivability of pathogenic bacteria inside a host cell also depends upon the stringent response demonstrated. Therefore, an understanding of the physiology of stringent conditions becomes very interesting in regulation of the growth and persistence of such invading pathogens.     

The suppression of defective translation by ppGpp and its role in the stringent response.

Amino acid starvation is shown to decrease the fidelity of translation in E. coli. When proteins are analyzed by two-dimensional gel electrophoresis, missense errors are detected as an unusual heterogeneity in their isoelectric points, while premature termination of protein synthesis can be recognized by a decreased relative rate of synthesis of higher molecular weight proteins and by the the accumulation of a complex group of new small polypeptides. The types of translational errors observed are amino acid-specific. For example, starvation of a rel- strain for histidine produces severe isoelectric point heterogeneity with little evidence of premature termination, while starvation for leucine has little effect on the isoelectric points, but produces a drastic decrease in the average molecular weight of the newly synthesized protein. These differences suggest codon-specific errors in reading the genetic code. In these rel- cells, the effect of amino acid starvation on the rates of synthesis of complete individual proteins is both protein- and amino acid-specific. For example, ribosomal protein L7/12, which lacks histidine, is made at a higher level during histidine starvation than during isoleucine or leucine starvation. This suggests that in rel- cells, the modulation of gene expression caused by the lack of a particular amino acid is, at least in part, a function of the abundance of that amino acid in particular proteins-that is, the response of rel- cells to starvation is consistent with the theory that the inhibition of protein synthesis and the accompanying increase in error frequency both result from low levels of the correct substrate. In marked contrast, virtually no starvation-induced translational errors are detected in a rel+ strain, and the response is not amino acid-specific. Varoius data strongly imply that in this rel+ strain, essentially all the changes caused by starvation are due to the accumulation of ppGpp, which independently reduces protein synthesis, thereby suppressing all the direct effects of amino acid limitation seen in rel- strains (where ppGpp does not accumulate upon starvation). A model is presented which describes how ppGpp might suppress the direct effects of starvation and avoid the loss of translational fidelity. In addition, the direct and specific effects of ppGpp on gene expression are examined independently of amino acid starvation.     

Synthesis of pppGpp by ribosomes from an Escherichia coli spoT mutant and the metabolic relationship between pppGpp and ppGpp.

Both ribosomes and a cell-free extract (S-30) prepared from an Escherichia coli spoT mutant catalyzed the synthesis of guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) as efficiently as did ribosomes and S-30 from a spoT+ strain. In both cases, the level of pppGpp reached its maximum before ppGpp maximally accumulated. pppGpp added to the ribosome system was rapidly converted to ppGpp. These results indicate that the spoT+ gene product may not have a direct role in the synthesis of pppGpp and that pppGpp is a precursor of ppGpp.     

Occurrence of the stringent response in Streptomyces sp. and its significance for the initiation of morphological and physiological differentiation

Streptomyces sp. MA406-A-1 produced formycin (a nucleoside antibiotic) in parallel with cell growth in a synthetic medium. When the synthetic medium was supplemented with 1% (w/v) Casamino acids, however, formycin was produced only after the end of exponential growth. The intracellular ppGpp pool increased gradually towards the end of exponential growth and was maximal at the beginning of formycin production. After shift down from Casamino acids medium to synthetic medium, the ppGpp pool increased immediately, while the GTP pool decreased; under such conditions, the ability to produce formycin increased eightfold. Relaxed (rel) mutants, the first isolated for a Streptomyces species, were found at high incidence (10%) among spontaneous thiopeptin-resistant isolates and had severely reduced abilities to accumulate ppGpp. These rel mutants also failed to produce formycin under the usual culture conditions and exhibited numerous pleiotropic effects such as an inability to produce melanin and an extended delay of aerial mycelium formation. Thus Streptomyces sp. exhibited a typical stringent response, and the response initiated (or was needed for) the induction of secondary metabolism. The response may have also participated in the initiation of aerial mycelium formation by decreasing the intracellular GTP pool.     

The temperature sensitive mutant 72c. II. Accumulation at high temperature of ppGpp and pppGpp in the presence of protein synthesis.

A heat sensitive mutant of E. coli has been analyzed. A shift to restrictive temperature leads to an accumulation of ppGpp and pppGpp in both the parental and the mutant strains (both are relA+). The pool of these compounds is shown to decrease with time after the temperature shift in the case of the parental strain, but remains at the same elevated level in the case of the mutant. The temperature shift of the mutant leads to an apparent reduction of stable RNA synthesis; this inhibition can be released by chloroamphenicol or tetracycline. Gross protein synthesis is more or less unaffected at restrictive temperature. In the parental strain little effect is seen on RNA and protein synthesis after the temperature shift. A relA derivative of the mutant does not show the same inhibition of RNA synthesis at high temperature. Sedimentation analysis suggests that mutant 70S ribosome are more stable, when exposed to a lowered Mg2+ concentration, than are 70S ribosomes from the parental strain. In addition, the relative amounts of the two forms of ribosomal protein S6, which can be obtained on DEAE chromatography (Held et al., 1973), are significantly changed in the mutant.     

Preparation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from Escherichia coli ribosomes

A means of preparative enzymatic synthesis of guanosine tetraphosphate (ppGpp), guanosine pentaphosphate (pppGpp), and related derivatives is deseribed. The Escherichia coli ribosomes can be recovered, stored, and used repeatedly as a source of synthetic activity. The procedure described affords a relatively simple means of synthesizing gram amounts of these nucleotides as well as some derivatives such as the β-γ methylenyl derivative of guanosine pentaphosphate (peppGpp).     

Guanine nucleotides in protein synthesis. Utilization of pppGpp and dGTP by initiation factor 2 and elongation factor Tu

Guanosine 5′-triphosphate, 3′-diphosphate (pppGpp), and dGTP support the initiation factor 2 (IF-2) and elongation factor Tu (EF-Tu) partial reactions of Escherichia coli protein synthesis. These natural analogs of GTP were as effective as GTP in supporting (1) IF-2-dependent binding of f-Met-tRNA to ribosomes, (2) IF-2-dependent formation of N-formylmethionylpuromycin, (3) EF-Tu-dependent binding of Phe-tRNA to a ribosome-polyuridylic acid-N-acetyl-Phe-tRNA complex, and (4) EF-Tu-dependent formation of N-acetyl-Phe-Phe-tRNA. GTP, pppGpp, and dGTP behaved similarly in time-course studies and across a broad concentration range with both enzymes. In addition, both GDP and guanosine 5′-diphosphate, 3′-diphosphate were found to be competitive inhibitors of both GTP and pppGpp in the IF-2- and EF-Tu-dependent reactions.     

Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: significance of the stringent response (ppGpp) and GTP content in relation to A factor.

I investigated the significance of the intracellular accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and of the coordinated decrease in the GTP pool for initiating morphological and physiological differentiation of Streptomyces griseus, a streptomycin-producing strain. In solid cultures, aerial mycelium formation was severely suppressed by the presence of excess nutrients. However, decoyinine, a specific inhibitor of GMP synthetase, enabled the cells to develop aerial mycelia in the suppressed cultures at concentrations which only partially inhibited growth. A factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone) added exogenously had no such effect. Decoyinine was also effective in initiating the formation of submerged spores in liquid culture. The ability to produce streptomycin did not increase but decreased drastically on the addition of decoyinine. This sharp decrease in streptomycin production was accompanied by a decrease in intracellular accumulation of ppGpp. A relaxed (rel) mutant was found among 25 thiopeptin-resistant isolates which developed spontaneously. The rel mutant had a severely reduced ability to accumulate ppGpp during a nutritional shift-down and also during postexponential growth and showed a less extensive decrease in the GTP pool than that in the rel+ parental strain. The rel mutant failed to induce the enzymes amidinotransferase and streptomycin kinase, which are essential for the biosynthesis of streptomycin. The abilities to form aerial mycelia and submerged spores were still retained, but the amounts were less, and for both the onset of development was markedly delayed. The decreased ability to produced submerged spores was largely restored by the addition of decoyinine. This was accompanied by an extensive GTP pool decrease. The rel mutant produced A factor normally, indicating that synthesis of A factor is controlled neither by ppGpp nor by GTP. Conversely, a mutant defective in A-factor synthesis accumulated as much ppGpp as did the parental strain. It was concluded that morphological differentiation of S. griseus results from a decrease in the pool of GTP, whereas physiological differentiation results from a more direct function of the rel gene product (ppGpp). It is also suggested that A factor may render the cell sensitive to receive and respond to the specified signal molecules, presumably ppGpp (for physiological differentiation) or GTP (for morphological differentiation).     

Glucose upshift of carbon-starved marine Vibrio sp. strain S14 causes amino acid starvation and induction of the stringent response.

The physiological status of carbon-starved cells of the marine Vibrio sp. strain S14 has been investigated by the analysis of their immediate response to carbon and energy sources. During the first minute after glucose addition to 48-h-starved cells, the pools of ATP and GTP increased rapidly, and the [ATP]/[ADP] ratio reached the level typical for growing cells within 4 min. The total rates of RNA and protein synthesis increased initially but were inhibited 4 to 5 min after glucose addition by the induction of the stringent response. A mutation in the relA gene abolished stringent control during the recovery and significantly prolonged the lag phase, before the starved cells regrew, after the addition of a single source of carbon. However, both the wild-type and the relA cells regrew without a significant lag phase when given glucose supplemented with amino acids. On the basis of these results, it is suggested that carbon-starved cells are deficient in amino acid biosynthesis and that ppGpp and the stringent response are involved in overcoming this deficiency, presumably by depressing the synthesis of amino acid biosynthetic enzymes. Furthermore, the data suggest that the starved cells primarily are starved for energy, and evidence is presented that the step-up in the rate of protein synthesis after refeeding is partially dependent on de novo RNA synthesis.     

Nitrofurantoin prompts the stringent response in Bacillus subtilis

Nitrofurantoin causes the stringent response in Bacillus subtilis. After exposure of a stringent strain to this drug, the intracellular concentrations of guanosine 3'-diphosphate 5'-diphosphate (ppGpp), guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and ATP increased, while that of GTP decreased. In a relaxed strain no accumulation of ppGpp or pppGpp was observed, but both GTP and ATP declined after the addition of nitrofurantoin. Protein synthesis was equally sensitive to nitrofurantoin in both the stringent and relaxed strains, but the drug inhibited RNA accumulation only in the stringent strain, not in the relaxed strain. Nitrofurantoin also caused the accumulation of ppGpp in Escherichia coli and Serratia marcescens.