Mutation in the rel gene of Sorangium cellulosum affects morphological and physiological differentiation

Interruption of the (p)ppGpp synthetase gene ( rel ) of Sorangium cellulosum So ce56 resulted in loss of ppGpp accumulation after norvaline treatment during exponential growth phase. The rel mutant failed to produce wild-type levels of the polyketides chivosazol and etnangien in production media. In wild-type cells expression of the chivosazol biosynthetic operon can be significantly increased by norvaline or α-methylglucoside. This induction does not occur in the rel mutant. The rel mutant also lost the capability to form multicellular fruiting bodies under nutrient starvation.     

Differential regulation of opposing RelMtb activities by the aminoacylation state of a tRNA.ribosome.mRNA.RelMtb complex

Rel(Mtb) of Mycobacterium tuberculosis is responsible for the intracellular regulation of (p)ppGpp and the consequent ability of the organism to survive long-term starvation, indicating a possible role in the pathogenesis of tuberculosis. Purified Rel(Mtb) is a dual-function enzyme carrying out ATP: GTP/GDP/ITP 3'-pyrophosphoryltransferase and (p)ppGpp 3'-pyrophosphohydrolase reactions. Here we show that in the absence of biological regulators, Rel(Mtb) simultaneously catalyzes both transferase and hydrolysis at the maximal rate for each reaction, indicating the existence of two distinct active sites. The differential regulation of the opposing activities of Rel(Mtb) is dependent on the ratio of uncharged to charged tRNA and the association of Rel(Mtb) with a complex containing tRNA, ribosomes, and mRNA. A 20-fold increase in the k(cat) and a 4-fold decrease in K(ATP) and K(GTP) from basal levels for transferase activity occur when Rel(Mtb) binds to a complex containing uncharged tRNA, ribosomes, and mRNA (Rel(Mtb) activating complex or RAC). The k(cat) for hydrolysis, however, is reduced 2-fold and K(m) for pppGpp increased 2-fold from basal levels in the presence of the Rel(Mtb) activating complex. The addition of charged tRNA to this complex has the opposite effect by inhibiting transferase activity and activating hydrolysis activity. Differential control of Rel(Mtb) gives the Mtb ribosomal complex a new regulatory role in controlling cellular metabolism in response to stringent growth conditions that may be present in the dormant Mtb lesion.     

Characterization of the stringent and relaxed responses of Streptococcus equisimilis.

The 739-codon rel(Seq) gene of Streptococcus equisimilis H46A is bifunctional, encoding a strong guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) and a weaker ribosome-independent ATP:GTP 3'-pyrophosphoryltransferase [(p)ppGpp synthetase]. To analyze the function of this gene, (p)ppGpp accumulation patterns as well as protein and RNA synthesis were compared during amino acid deprivation and glucose exhaustion between the wild type and an insertion mutant carrying a rel(Seq) gene disrupted at codon 216. We found that under normal conditions, both strains contained basal levels of (p)ppGpp. Amino acid deprivation imposed by pseudomonic acid or isoleucine hydroxamate triggered a rel(Seq)-dependent stringent response characterized by rapid (p)ppGpp accumulation at the expense of GTP and abrupt cessation of net RNA accumulation in the wild type but not in the mutant. Tetracycline added to block (p)ppGpp synthesis caused the accumulated (p)ppGpp to degrade rapidly, with a concomitant increase of the GTP pool (decay constant of ppGpp, approximately 0.7 min(-1)). Simultaneous addition of pseudomonic acid and tetracycline to mimic a relaxed response caused wild-type RNA synthesis to proceed at rates approximating those seen under either condition in the mutant. Glucose exhaustion provoked the (p)ppGpp accumulation response in both the wild type and the rel(Seq) insertion mutant, consistent with the block of net RNA accumulation in both strains. Although the source of (p)ppGpp synthesis during glucose exhaustion remains to be determined, these findings reinforce the idea entertained previously that rel(Seq) fulfils functions that reside separately in the paralogous reL4 and spoT genes of Escherichia coli. Analysis of (p)ppGpp accumulation patterns was complicated by finding an unknown phosphorylated compound that comigrated with ppGpp under two standard thin-layer chromatography conditions. Unlike ppGpp, this compound did not adsorb to charcoal and did not accumulate appreciably during isoleucine deprivation. Like ppGpp, the unknown compound did accumulate during energy source starvation.     

Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis.

We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants.     

Functional regulation of the opposing (p)ppGpp synthetase/hydrolase activities of RelMtb from Mycobacterium tuberculosis

The dual-function Rel(Mtb) protein from Mycobacterium tuberculosis catalyzes both the synthesis and hydrolysis of (p)ppGpp, the effector of the stringent response. In our previous work [Avarbock, D., Avarbock, A., and Rubin, H. (2000) Biochemistry 39, 11640], we presented evidence that the Rel(Mtb) protein might catalyze its two opposing reactions at distinct active sites. In the study presented here, we purified and characterized fragments of the 738-amino acid Rel(Mtb) protein and confirmed the hypothesis that amino acid fragment 1-394 contains both synthesis and hydrolysis activities, amino acid fragment 87-394 contains only (p)ppGpp synthesis activity, and amino acid fragment 1-181 contains only (p)ppGpp hydrolysis activity. Mutation of specific residues within fragment 1-394 results in the loss of synthetic activity and retention of hydrolysis (G241E and H344Y) or loss of hydrolytic activity with retention of synthesis (H80A and D81A). The C-terminally cleaved Rel(Mtb) fragment proteins have basal activities similar to that of full-length Rel(Mtb), but are no longer regulated by the previously described Rel(Mtb) activating complex (RAC). Residues within the C-terminus of Rel(Mtb) (D632A and C633A) are shown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicates Rel(Mtb) forms trimers and removal of the C-terminus results in monomers. The C-terminal deletion, 1-394, which exists as a mixture of monomers and trimers, will dissociate from the trimer state upon the addition of substrate. Furthermore, the trimer state of fragment 1-394 appears to be a catalytically less efficient state than the monomer state.     

Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT.

The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp. strain S14 is subjected to carbon and energy starvation. By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp. strain S14. The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp. S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins. However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation. We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp. strain S14. It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp. S14 spoT mutant strain (csrS1). Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival. On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell. This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied. The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.     

Lowering GTP Level Increases Survival of Amino Acid Starvation but Slows Growth Rate for Bacillus subtilis Cells Lacking (p)ppGpp

Bacterial cells sense external nutrient availability to regulate macromolecular synthesis and consequently their growth. In the Gram-positive bacterium Bacillus subtilis, the starvation-inducible nucleotide (p)ppGpp negatively regulates GTP levels, both to resist nutritional stress and to maintain GTP homeostasis during growth. Here, we quantitatively investigated the relationship between GTP level, survival of amino acid starvation, and growth rate when GTP synthesis is uncoupled from its major homeostatic regulator, (p)ppGpp. We analyzed growth and nucleotide levels in cells that lack (p)ppGpp and found that their survival of treatment with a nonfunctional amino acid analog negatively correlates with both growth rate and GTP level. Manipulation of GTP levels modulates the exponential growth rate of these cells in a positive dose-dependent manner, such that increasing the GTP level increases growth rate. However, accumulation of GTP levels above a threshold inhibits growth, suggesting a toxic effect. Strikingly, adenine counteracts GTP stress by preventing GTP accumulation in cells lacking (p)ppGpp. Our results emphasize the importance of maintaining appropriate levels of GTP to maximize growth: cells can survive amino acid starvation by decreasing GTP level, which comes at a cost to growth, while (p)ppGpp enables rapid adjustment to nutritional stress by adjusting GTP level, thus maximizing fitness.     

Cloning and characterization of a bifunctional RelA/SpoT homologue from Mycobacterium tuberculosis.

A 2.2kb relA/spoT homologue was isolated from Mycobacterium tuberculosis (Mtb) genomic DNA by PCR-amplification. The Mtb gene encodes a protein of 738 amino acid residues, and is flanked upstream by an ORF that is highly similar to the apt gene, and downstream by an ORF that is highly similar to the cypH gene. This dual function Mtb homologue belongs to the relA/spoT family of genes that mediate the stringent response by regulating the synthesis and degradation of guanosine 3',5'-bis(diphosphate) (ppGpp) and pppGpp. In vitro biochemical data indicate that purified RelMtb is a ribosome- and tRNA-independent ATP:GTP/GDP/ITP 3'-pyrophosphoryltransferase. Additionally, purified RelMtb is an Mn2+-dependent, ribosome and tRNA-independent, (p)ppGpp 3'-pyrophosphohydrolase. These reactions were also assessed in vivo in E. coli deleted in both the relA and spoT genes, which generates a (p)ppGpp0 phenotype. RelMtb can suppress this phenotype and can generate more (p)ppGpp than relA in the wild type E. coli control.     

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.     

Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.     

Differential stringent responses of Streptomyces coelicolor M600 to starvation of specific nutrients

This study focused on the involvement of the unusual nucleotide (p)ppGpp, a stringent factor, during the morphological and physiological differentiation of Streptomyces coelicolor. Two genes, relA and rshA, were disrupted to demonstrate the roles of the stringent factor in the differentiation. The intracellular concentration of (p)ppGpp in the wild-type (M600) and disrupted mutants was measured in relation to the intentional starvation of a specific nutrient such as carbon, nitrogen, and phosphate or the in situ depletion of nutrients in a batch culture. As a result, it was found that the morphological characteristic of the deltarelA mutant was a bld phenotype forming condensed mycelia, whereas the deltarshA mutant grew fast-forming spores and straightforward mycelia. In both mutants, the production of actinorhodin (Act) was completely abolished, yet the undecylprodigiosin (Red) production was increased. Intracellular (p)ppGpp was detected in the deltarelA mutant in the case of limited phosphate, yet not with limited carbon or nitrogen sources. In contrast, (p)ppGpp was produced in the deltarshA mutant under limited carbon and nitrogen conditions. Therefore, (p)ppGpp in S. coelicolor was found to be selectively regulated by either the RelA or RshA protein, which was differentially expressed in response to the specific nutrient limitation. These results were also supported by the in situ ppGpp production during a batch culture. Furthermore, it is suggested that RelA and RshA are bifunctional proteins that possess the ability to both synthesize and hydrolyze (p)ppGpp.     

Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC27064

The effects of growth rate and nutrient uptake rate on the production of cephamycin C were determined in the parental strain, deltarelA mutant, and deltarsh null mutant of S. clavuligerus. Production of cephamycin C was inversely related to mycelium growth and the phosphate feed rate was more critical for the production of cephamycin C. On the contrary, the production of cephamycin C was completely abolished in the deltarelA mutant, but not in deltarsh mutant. The changes in the cephamycin C production by disruption of the relA and rsh genes are presumably associated with the consequent ability of the mutants to accumulate (p)ppGpp under nutrient starvation. Therefore, it is concluded that the stringent response of S. clavuligerus to starvation for nutrients is governed mainly by RelA rather than Rsh and that the response is more apparently regulated by the limitation of phosphate.     

Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus equisimilis

Catalytic and regulatory domains of the Rel/Spo homolog of Streptococcus equisimilis affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared. Two major domains of the 739-residue Rel(Seq) protein are defined by limited proteolytic digestion. In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3'-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn(2+)-dependent 3'-pyrophosphohydrolase. Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein. Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis. Cellular (p)ppGpp accumulation behavior is consistent with these changes. The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro. These assignments can also apply to RelA and SpoT. The ability of Rel(Seq) to mediate (p)ppGpp accumulation during amino acid starvation in S. equisimilis is absent when the protein is expressed ectopically in Escherichia coli. Fusing the N-terminal half of Rel(Seq) with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in E. coli by inhibiting unregulated degradation and restoring regulated synthetic activity. Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins.