Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain

The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in relA⁺ cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.     

Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli

Amino acid-deprived rplK (previously known as relC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative of rplK encoding L11 lacking the N-terminal 36 amino acids (designated 'L11) was constructed. Bacteria overexpressing 'L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed 'L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.     

The relA/spoT-Homologous Gene in Streptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.     

Dependence of RelA-mediated (p)ppGpp formation on tRNA identity

The bacterial stringent response is a cellular response to amino acid limitations and is characterized by the accumulation of the alarmone polyphosphate guanosine ((p)ppGpp). A key molecular event leading to (p)ppGpp synthesis is the binding of a deacylated tRNA to the vacant A-Site of a ribosome. The resulting ribosomal complex is recognized by and activates RelA, the (p)ppGpp synthetase. Activated RelA catalyzes (p)ppGpp formation until the deacylated tRNA passively dissociates from the ribosomal A-Site. In this report, we have investigated a novel role for the identity of A-Site bound tRNA in RelA-mediated (p)ppGpp synthesis. A comparison in the stimulation of RelA activity was made using ribosome complexes with either a tightly or weakly binding deacylated tRNA occupying the A-Site. In vitro analysis reveals that ribosome complexes formed with tight binding tRNA(Val) stimulate RelA activity at lower concentrations than that required for ribosome complexes formed with the weaker binding tRNA(Phe). The data suggest that the recovery from the stringent response may be dependent on the identity of the amino acid that was initially limiting for the bacteria.     

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.     

魔斑合成酶的分离、鉴定及功能分析

细菌响应营养饥饿或环境胁迫的反应称为严谨反应.本文采用Native-PAGE技术从毒死蜱胁迫下的Klebsiella sp.CPK菌株全细胞蛋白中分离得到了1种特异蛋白,该蛋白通过TOF-MS测序推测为魔斑合成酶RelA.对该蛋白编码基因进行PCR扩增,并利用ORF Finder,showorf等生物信息学软件对其开放性阅读框架进行鉴定,获得了1个全长为2 238bp的完整relA基因.序列及系统发育分析表明,Klebsiella sp. CPK菌株的RelA蛋白与E.coli RelA蛋白的同源性为92%,但与双功能的Rel-SpoT蛋白同源性却比较低.由此推测,Klebsiella sp. CPK RelA蛋白可能只有魔斑合成酶活性.另外,对relA基因进行功能互补分析表明,该基因编码的特异蛋白具有魔斑合成酶活性,从而证明了Klebsiella sp. CPK菌株在毒死蜱胁迫下能够产生典型的严谨反应。     

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.     

The stringent response is required for amino acid and nitrate utilization, nod factor regulation, nodulation, and nitrogen fixation in Rhizobium etli

A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a DeltarelA DeltaspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Omega element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-beta-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.     

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.     

Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli.

The penicillin tolerance exhibited by amino acid-deprived Escherichia coli has been previously proposed to be a consequence of the stringent response. Evidence indicating that penicillin tolerance is directly attributable to guanosine 3',5'-bispyrophosphate (ppGpp) overproduction and not to some other effect of amino acid deprivation is now presented. Accumulation of ppGpp in the absence of amino acid deprivation was achieved by the controlled overexpression of the cloned relA gene, which encodes ppGpp synthetase I. The overproduction of ppGpp resulted in the inhibition of both peptidoglycan and phospholipid synthesis and in penicillin tolerance. The minimum concentration of ppGpp required to establish these phenomena was determined to be 870 pmol per mg (dry weight) of cells. This represented about 70% of the maximum level of ppGpp accumulated during the stringent response. Penicillin tolerance and the inhibition of peptidoglycan synthesis were both suppressed when ppGpp accumulation was prevented by treatment with chloramphenicol, an inhibitor of ppGpp synthetase I activation. Glycerol-3-phosphate acyltransferase, the product of plsB, was recently identified as the main site of ppGpp inhibition in phospholipid synthesis (R. J. Health, S. Jackowski, and C. O. Rock, J. Biol. Chem. 269:26584-26590, 1994). The overexpression of the cloned plsB gene reversed the penicillin tolerance conferred by ppGpp accumulation. This result supports previous observations indicating that the membrane-associated events in peptidoglycan metabolism were dependent on ongoing phospholipid synthesis. Interestingly, treatment with beta-lactam antibiotics by itself induced ppGpp accumulation, but the maximum levels attained were insufficient to confer penicillin tolerance.     

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.     

Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli

The most widely studied "relaxed" mutant of the relA locus, the relA1 allele, is shown here to consist of an IS2 insertion between the 85th and 86th codons of the otherwise wild-type relA structural gene, which normally encodes a 743-amino acid (84 kDa) protein. The RelA protein is a ribosome-dependent ATP:GTP (GDP) pyrophosphoryltransferase that is activated during the stringent response to amino acid starvation and thereby occasions the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp). We propose that the IS2 insertion functionally splits the RelA protein into two (alpha and beta) peptide fragments which can complement each other in trans to yield residual ppGpp synthetic activity; neither fragment shows this activity when expressed alone. Cell strains with a single copy relA null allele show physiological behavior that is much the same as relA1 mutant strains. Both relA1 and relA null strains accumulate ppGpp during glucose starvation and do not accumulate ppGpp during the stringent response. The presence of ppGpp in verifiable relA null strains is interpreted as unequivocal evidence for an alternate route of ppGpp synthesis that exists in addition to the relA-dependent reaction.     

Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies

To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the alpha-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between alpha-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1-593 and 704-719) bound (125)I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1-601 and 650-719) and greater than that of dimeric mIR.Fn0 (amino acids 1-601 and 704-719) and monomeric IR473.CT (amino acids 1-473 and 704-719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound (125)I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471-593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.