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.     

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.     

Nutritional control of elongation of DNA replication by (p)ppGpp

DNA replication is highly regulated in most organisms. Although much research has focused on mechanisms that regulate initiation of replication, mechanisms that regulate elongation of replication are less well understood. We characterized a mechanism that regulates replication elongation in the bacterium Bacillus subtilis. Replication elongation was inhibited within minutes after amino acid starvation, regardless of where the replication forks were located on the chromosome. We found that small nucleotides ppGpp and pppGpp, which are induced upon starvation, appeared to inhibit replication directly by inhibiting primase, an essential component of the replication machinery. The replication forks arrested with (p)ppGpp did not recruit the recombination protein RecA, indicating that the forks are not disrupted. (p)ppGpp appear to be part of a surveillance mechanism that links nutrient availability to replication by rapidly inhibiting replication in starved cells, thereby preventing replication-fork disruption. This control may be important for cells to maintain genomic integrity.     

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.     

SpoT regulates DnaA stability and initiation of DNA replication in carbon-starved Caulobacter crescentus

Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response of C. crescentus swarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol. 55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.     

ppGpp-mediated regulation of DNA replication and cell division in Escherichia coli

ppGpp serves as an alarmon in prokaryotes, distributing and coordinating different cellular processes according to the nutritional potential of the growth medium. This work is interpreted as favoring the view that, in addition to its previously documented role in regulating the rate of ribosome synthesis [4], ppGpp participates in coordinating DNA replication and cell division. We studied the effects of ppGpp on the cell division cycle, using cells containing plasmid pSM11 that codes for the 55-kDa truncated RelA protein under the inducible Ptac promoter. In this system it was found that the rate of initiation of new rounds of DNA replication is inversely correlated with the intracellular level of ppGpp. Furthermore, ppGpp levels similar to those found during the activation of stringent control inhibited replication initiation, in a manner comparable to that resulting from inhibition of protein synthesis by amino acid starvation or by chloramphenicol addition. However, in contrast to chloramphenicol treatment, elevated ppGpp levels did not block septum formation, and, in fact, there is some evidence for enhanced septation. As a result, the residual cell division following elevation in ppGpp levels was higher than after chloramphenicol treatment, resulting in cells with a size similar to that of stationary phase cells.     

Replication and expression of plasmid pBR 322 during discontinuous culture of a stringent and relaxed Escherichia coli strain

The E. coli strains CP78 and CP79 carrying the plasmid pBR 322 display similar growth kinetics in discontinuous culture. During limitation of amino acids the stringent strain CP78 is able to synthesize guanosine-5'diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3' diphosphate, but the relaxed strain can not produce highly phosphorylated guanosine nucleotides. During the logarithmic phase of growth both strains contain similar amounts of plasmid DNA. During amino acid starvation plasmid DNA is amplified in the relaxed strain only, whereas in the stringent strain the plasmid content per cell remains constant. In stationary phase cells of CP78 a higher activity of plasmid-coded beta-lactamase than in CP79 cells was detected. Furthermore, remarkable differences between both strains were observed in the composition of proteins derived from the periplasmic fraction and separated by polyacrylamide gel electrophoresis. Our results might indicate a negative control of pBR 322 DNA replication by ppGpp during amino acid starvation.     

(p)ppGpp——“魔斑”核苷酸在细菌中的研究进展

鸟苷四磷酸（guanosine tetraphosphate,ppGpp）/鸟苷五磷酸（guanosine pentaphosphate,pppGpp）是细菌严谨反应的信号分子,其合成和水解由Rel/SpoT同系物（RelA/SpoT homologue,RSH）家族的蛋白质合成和水解活性控制。（p）ppGpp介导的严谨反应能够提高细菌对营养匮乏的适应能力和抗生素抗性。近年来发现（p）ppGpp与细菌生长和细胞分裂、抗生素合成等都密切相关,是细胞内重要的全局调控因子。（p）ppGpp在细菌细胞中有许多靶点,使其可以调节DNA复制、转录、细胞周期、核糖体生物合成以及抗生素合成基因簇的表达。然而,（p）ppGpp如何控制转录和其他代谢过程取决于细菌种类,并在不同的微生物中通过不同的机制调节相同的过程。因此,本文通过综述（p）ppGpp的合成/水解酶的种类和调节机制,（p）ppGpp对微生物代谢调控机制、对细胞周期的影响机制,以及（p）ppGpp对抗生素合成和耐受性的调控机制,为细菌耐药性研究和细胞生理学研究奠定基础。     

A checkpoint involving RTP, the replication terminator protein, arrests replication downstream of the origin during the Stringent Response in Bacillus subtilis

Regulation of DNA replication in Bacillus subtilis involves a post-initiation mechanism which is subject to control by the Stringent System, an essential regulatory network, mediated by the alarmone, ppGpp. In detailed studies using DNA-DNA hybridization procedures, we have now shown that, following the induction of the Stringent Response, replication is blocked downstream of the origin, on the left, close to the hut marker (-175 kb) and on the right, beyond the soft10 marker (+199 kb). In addition, we provide evidence that inhibition of replication under these conditions requires the replication terminator protein (RTP). In a mutant lacking RTP, a protein normally involved in termination of chromosomal replication through recognition of specific terminator sequences, replication continues past the sites normally blocked by the Stringent Response. These data strengthen the argument that this second level of control of DNA replication occurs at specific sites, the Stringent Terminus (STer) sites, either side of oriC Such sites are presumably related to the sequence involved in RTP recognition at the terminus, terC. We propose that the binding of RTP must be modulated, perhaps through the action of ppGpp, to recognize post-initiation control sequences during the Stringent Response, in order to block replisome movement. This, therefore, acts as a checkpoint in chromosome elongation.