Correlation between RNA synthesis and basal level guanosine 5'-diphosphate 3'-diphosphate in relaxed mutants of Escherichia coli

Through the use of a new nucleotide extraction procedure, we had previously shown that relaxed mutants of Escherichia coli exhibit a unique response to amino acid starvation (Lagosky, P. A., and Chang, F. N. (1980) J. Bacteriol. 144, 499-508). The basal level amounts of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA and phenotypically relaxed relA+ rplK (relC) strains were shown to decrease at the onset of amino acid limitation and to remain severely depressed throughout the course of the starvation. Upon resupplementation of amino acid-starved relaxed mutants, the production of ppGpp resumes and results in the temporary overaccumulation of this nucleotide beyond its original basal level amount. We now show that the basal level ppGpp content of relaxed bacteria, as well as its subsequent fluctuations in response to amino acid starvation, is inversely correlated with the initial rates of RNA synthesis in these strains. The ability of ppGpp to control the rate of protein synthesis in relA mutants was also examined. It was observed that ppGpp had no apparent direct effect on the initial rates of protein synthesis in relA mutants. The constant inverse correlation which exists between ppGpp content in relA mutants, and their rates of RNa synthesis provide evidence which indicates that basal level ppGpp synthesis has definite physiological significance. It also suggests that the synthesis of basal level ppGpp might be an absolute requirement needed for normal bacterial growth.     

The bacterial stringent response, conserved in chloroplasts, controls plant fertilization

The chloroplast, an essential organelle for plants, performs a wide variety of metabolic processes for host cells, which include photosynthesis as well as amino acid and fatty acid biosynthesis. The organelle conserves many bacterial systems in its functions, implicating its origin from symbiosis of a photosynthetic bacterium. In bacterial cells, the stringent response acts as a global regulatory system for gene expression mediated by a small nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), that is necessary for cell adaptation to diverse environmental stimuli such as amino acid starvation. Recent studies indicated that proteins similar to the bacterial ppGpp synthase/hydrolyase are conserved in plants, although their precise roles are not known. Here we show that the stringent response in chloroplasts is crucial for normal plant fertilization. Specifically, one of the Arabidopsis ppGpp synthase homologs, CRSH (Ca(2+)-activated RelA/SpoT homolog), exhibits calcium-dependent ppGpp synthesis activity in vitro, and is localized in chloroplasts in vivo. A knockdown mutation of CRSH in Arabidopsis results in a significant reduction in silique size and seed production, indicating that plant reproduction is under the control of chloroplast function through a ppGpp-mediated stringent response.     

Occurrence of the regulatory nucleotides ppGpp and pppGpp following induction of the stringent response in staphylococci.

The stringent response in Escherichia coli and many other organisms is regulated by the nucleotides ppGpp and pppGpp. We show here for the first time that at least six staphylococcal species also synthesize ppGpp and pppGpp upon induction of the stringent response by mupirocin. Spots corresponding to ppGpp and pppGpp on thin-layer chromatograms suggest that pppGpp is the principal regulatory nucleotide synthesized by staphylococci in response to mupirocin, rather than ppGpp as in E. coli.     

Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis

In nearly all bacterial species examined so far, amino acid starvation triggers the rapid accumulation of the nucleotide second messenger (p)ppGpp, the effector of the stringent response. While for years the enzymes involved in (p)ppGpp metabolism and the significance of (p)ppGpp accumulation to stress survival were considered well defined, a recent surge of interest in the field has uncovered an unanticipated level of diversity in how bacteria metabolize and utilize (p)ppGpp to rapidly synchronize a variety of biological processes important for growth and stress survival. In addition to the classic activation of the stringent response, it has become evident that (p)ppGpp exerts differential effects on cell physiology in an incremental manner rather than simply acting as a biphasic switch that controls growth or stasis. Of particular interest is the intimate relationship of (p)ppGpp with persister cell formation and virulence, which has spurred the pursuit of (p)ppGpp inhibitors as a means to control recalcitrant infections. Here, we present an overview of the enzymes responsible for (p)ppGpp metabolism, elaborate on the intricacies that link basal production of (p)ppGpp to bacterial homeostasis, and discuss the implications of targeting (p)ppGpp synthesis as a means to disrupt long-term bacterial survival strategies.     

The Alarmone (p)ppGpp Regulates Primer Extension by Bacterial Primase

Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis. Bacterial primase activity can be regulated by the starvation-inducible nucleotide (p)ppGpp. This regulation contributes to a timely inhibition of DNA replication upon amino acid starvation in the Gram-positive bacterium Bacillus subtilis. Here, we characterize the effect of (p)ppGpp on B. subtilis DnaG primase activity in vitro. Using a single-nucleotide resolution primase assay, we dissected the effect of ppGpp on the initiation, extension, and fidelity of B. subtilis primase. We found that ppGpp has a mild effect on initiation, but strongly inhibits primer extension and reduces primase processivity, promoting termination of primer extension. High (p)ppGpp concentration, together with low GTP concentration, additively inhibit primase activity. This explains the strong inhibition of replication elongation during starvation which induces high levels of (p)ppGpp and depletion of GTP in B. subtilis. Finally, we found that lowering GTP concentration results in mismatches in primer base pairing that allow priming readthrough, and that ppGpp reduces readthrough to protect priming fidelity. These results highlight the importance of (p)ppGpp in protecting replisome integrity and genome stability in fluctuating nucleotide concentrations upon onset of environmental stress.     

G-protein control of the ribosome-associated stress response protein SpoT

The bacterial response to stress is controlled by two proteins, RelA and SpoT. RelA generates the alarmone (p)ppGpp under amino acid starvation, whereas SpoT is responsible for (p)ppGpp hydrolysis and for synthesis of (p)ppGpp under a variety of cellular stress conditions. It is widely accepted that RelA is associated with translating ribosomes. The cellular location of SpoT, however, has been controversial. SpoT physically interacts with the ribosome-associated GTPase CgtA, and we show here that, under an optimized salt condition, SpoT is also associated with a pre-50S particle. Analysis of spoT and cgtA mutants and strains overexpressing CgtA suggests that the ribosome associations of SpoT and CgtA are mutually independent. The steady-state level of (p)ppGpp is increased in a cgtA mutant, but the accumulation of (p)ppGpp during amino acid starvation is not affected, providing strong evidence that CgtA regulates the (p)ppGpp level during exponential growth but not during the stringent response. We show that CgtA is not associated with pre-50S particles during amino acid starvation, indicating that under these conditions in which (p)ppGpp accumulates, CgtA is not bound either to the pre-50S particle or to SpoT. We propose that, in addition to its role as a 50S assembly factor, CgtA promotes SpoT (p)ppGpp degradation activity on the ribosome and that the loss of CgtA from the ribosome is necessary for maximal (p)ppGpp accumulation under stress conditions. Intriguingly, we found that in the absence of spoT and relA, cgtA is still an essential gene in Escherichia coli.     

Role of peptide chain elongation factor G in guanosine 5'-diphosphate 3'-diphosphate synthesis.

In a wild-type strain (relA+) of Escherichia coli, starvation of amino acid led to an immediate cessation of the synthesis of stable ribonucleic acids, together with the accumulation of an unusual nucleotide, guanosine 5'-diphosphate 3'-diphosphate, commonly known as ppGpp. This compound also accumulated during heat shock. When temperature-sensitive protein synthesis elongation factor G (EF-G) was introduced into E. coli NF859, a relA+ strain, the synthesis of ppGpp was reduced to approximately one-half that of wild-type EF-G+ cells at a nonpermissive temperature of 40 degrees C. Furthermore, fusidic acid, an inhibitor of protein synthesis which specifically inactivates EF-G, prevented any accumulation of ppGpp during the heat shock. We suggest that a functional EF-G protein is necessary for ppGpp accumulation under temperature shift conditions, possibly by mediating changes in the function of another protein, the relA gene product. However, EF-G is probably not required for the synthesis of ppGpp during the stringent response, since its inactivation did not prevent ppGpp accumulation during amino acid starvation.     

ppGpp-Dependent Negative Control of DNA Replication of Shiga Toxin-Converting Bacteriophages in Escherichia coli

The pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains depends on the production of Shiga toxins that are encoded on lambdoid prophages. Effective production of these toxins requires prophage induction and subsequent phage replication. Previous reports indicated that lytic development of Shiga toxin-converting bacteriophages is inhibited in amino acid-starved bacteria. However, those studies demonstrated that inhibition of both phage-derived plasmid replication and production of progeny virions occurred during the stringent as well as the relaxed response to amino acid starvation, i.e., in the presence as well as the absence of high levels of ppGpp, an alarmone of the stringent response. Therefore, we asked whether ppGpp influences DNA replication and lytic development of Shiga toxin-converting bacteriophages. Lytic development of 5 such bacteriophages was tested in an E. coli wild-type strain and an isogenic mutant that does not produce ppGpp (ppGpp⁰). In the absence of ppGpp, production of progeny phages was significantly (in the range of an order of magnitude) more efficient than in wild-type cells. Such effects were observed in infected bacteria as well as after prophage induction. All tested bacteriophages formed considerably larger plaques on lawns formed by ppGpp⁰ bacteria than on those formed by wild-type E. coli. The efficiency of synthesis of phage DNA and relative amount of lambdoid plasmid DNA were increased in cells devoid of ppGpp relative to bacteria containing a basal level of this nucleotide. We conclude that ppGpp negatively influences the lytic development of Shiga toxin-converting bacteriophages and that phage DNA replication efficiency is limited by the stringent control alarmone.