The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life

RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the "stringent response" to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.     

Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein-SpoT interaction

Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.     

Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis

The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the ‘alarmones’ (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5′,3′-dibisphosphate guanosine) with an EC₅₀ of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia.     

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.     

Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation

The spoT gene of Escherichia coli encodes a guanosine 3′,5′-bis(diphosphate) 3′-pyrophosphohydrolase (ppGppase) as well as an apparent guanosine 3′,5′-bis(diphosphate) synthetase (designated PSII). To determine the regions of the SpoT protein that are required for these two competing activities, we analysed plasmid-borne deletion mutations for their ability to complement chromosomal mutations defective in each activity. We found that a region containing the first 203 amino acids of the 702-amino-acid SpoT protein was sufficient for ppGppase activity while an overlapping region containing residues 67–374 was sufficient for PSII activity. These data indicate that the catalytic sites involved in the two activities are separate but closely linked in the primary sequence of the SpoT protein. A ppGppase-defective Δ1–58 deletion mutant strain failed to synthesize ppGpp in response to nutrient limitation, also supporting the notion that PSII activity from wild-type SpoT does not increase in response to nutrient limitation. Using a strain lacking PSII activity but retaining ppGppase activity, we determined the contribution of the RelA protein (ppGpp synthetase I, PSI) to ppGpp synthesis following glucose starvation. We found that the RelA protein activity accounts for the initial burst of ppGpp synthesis at the onset of glucose starvation but that this source of synthesis is absent when amino acids are present during glucose starvation.     

Plant mechanism of an adaptive stress response homologous to bacterial stringent response

All living organisms possess adaptive responses to environmental stresses that are essential to ensuring cell survival. One of them is the stringent response, initially discovered forty years ago in the gram-negative model organism E. coli. Recently plant homologues to the bacterial relA/spoT genes were identified (RSH genes--RelA/SpoT Homologues). Also the products of rsh proteins activity--(p)ppGpp were identified in the chloroplasts of plant cells. Levels of ppGpp increased markedly when plants were subjected to some biotic and abiotic stresses. Elevation of ppGpp levels was elicited also by treatment with plant hormones. What is more--in vitro, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp. It is supposed that plant stringent response is a conserve stress-response pathway possibly operating via regulation of chloroplast gene expression and, thus, the regulation of plastid metabolism.     

Phylogenetic analysis of proteins involved in the stringent response in plant cells

The nucleotide (p)ppGpp is a second messenger that controls the stringent response in bacteria. The stringent response modifies expression of a large number of genes and metabolic processes and allows bacteria to survive under fluctuating environmental conditions. Recent genome sequencing analyses have revealed that genes responsible for the stringent response are also found in plants. These include (p)ppGpp synthases and hydrolases, RelA/SpoT homologs (RSHs), and the pppGpp-specific phosphatase GppA/Ppx. However, phylogenetic relationship between enzymes involved in bacterial and plant stringent responses is as yet generally unclear. Here, we investigated the origin and evolution of genes involved in the stringent response in plants. Phylogenetic analysis and primary structures of RSH homologs from different plant phyla (including Embryophyta, Charophyta, Chlorophyta, Rhodophyta and Glaucophyta) indicate that RSH gene families were introduced into plant cells by at least two independent lateral gene transfers from the bacterial Deinococcus-Thermus phylum and an unidentified bacterial phylum; alternatively, they were introduced into a proto-plant cell by a lateral gene transfer from the endosymbiotic cyanobacterium followed by gene loss of an ancestral RSH gene in the cyanobacterial linage. Phylogenetic analysis of gppA/ppx families indicated that plant gppA/ppx homologs form an individual cluster in the phylogenetic tree, and show a sister relationship with some bacterial gppA/ppx homologs. Although RSHs contain a plastidial transit peptide at the N terminus, GppA/Ppx homologs do not, suggesting that plant GppA/Ppx homologs function in the cytosol. These results reveal that a proto-plant cell obtained genes for the stringent response by lateral gene transfer events from different bacterial phyla and have utilized them to control metabolism in plastids and the cytosol.     

(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。     

Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis

Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA-SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins ( approximately 25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.     

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.