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.     

Temperature-sensitive growth and decreased thermotolerance associated with relA mutations in Escherichia coli

The relA gene of Escherichia coli encodes guanosine 3',5'-bispyrophosphate (ppGpp) synthetase I, a ribosome-associated enzyme that is activated during amino acid starvation. The stringent response is thought to be mediated by ppGpp. Mutations in relA are known to result in pleiotropic phenotypes. We now report that three different relA mutant alleles, relA1, relA2, and relA251::kan, conferred temperature-sensitive phenotypes, as demonstrated by reduced plating efficiencies on nutrient agar (Difco) or on Davis minimal agar (Difco) at temperatures above 41 degrees C. The relA-mediated temperature sensitivity was osmoremedial and could be completely suppressed, for example, by the addition of NaCl to the medium at a concentration of 0.3 M. The temperature sensitivities of the relA mutants were associated with decreased thermotolerance; e.g., relA mutants lost viability at 42 degrees C, a temperature that is normally nonlethal. The spoT gene encodes a bifunctional enzyme possessing ppGpp synthetase and ppGpp pyrophosphohydrolase activities. The introduction of the spoT207::cat allele into a strain bearing the relA251::kan mutation completely abolished ppGpp synthesis. This ppGpp null mutant was even more temperature sensitive than the strain carrying the relA251::kan mutation alone. The relA-mediated thermosensitivity was suppressed by certain mutant alleles of rpoB (encoding the beta subunit of RNA polymerase) and spoT that have been previously reported to suppress other phenotypic characteristics conferred by relA mutations. Collectively, these results suggest that ppGpp may be required in some way for the expression of genes involved in thermotolerance.     

Synthesis of pppGpp by ribosomes from an Escherichia coli spoT mutant and the metabolic relationship between pppGpp and ppGpp.

Both ribosomes and a cell-free extract (S-30) prepared from an Escherichia coli spoT mutant catalyzed the synthesis of guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) as efficiently as did ribosomes and S-30 from a spoT+ strain. In both cases, the level of pppGpp reached its maximum before ppGpp maximally accumulated. pppGpp added to the ribosome system was rapidly converted to ppGpp. These results indicate that the spoT+ gene product may not have a direct role in the synthesis of pppGpp and that pppGpp is a precursor of ppGpp.     

Role of the spoT gene product and manganese ion in the metabolism of guanosine 5'-diphosphate 3'-diphosphate in Escherichia coli.

Addition of divalent ion chelating agents picolinic acid, 1,10-phenanthroline, or quinoline-2-carboxylic acid to wild type, relA, or relX, but not spoT strains of Escherichia coli increases the levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Poorly chelating analogs of these agents and a larger and more highly charged chelating agent, ethylene glycol bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid are ineffective. Mn2+ reverses the increase in ppGpp. The increase in ppGpp in wild type cells can be explained by an inhibition of degradation. In spoT cells the response is more complex; ppGpp does not increase although degradation is completely inhibited. The lack of increase in spoT cells suggests a role for spoT in synthesis of ppGpp in addition to its known role in degradation. Growth of both spoT+ and spoT cells is inhibited following chelator addition. This suggests that growth inhibition is through a mechanism not directly involving ppGpp. The results of this study provide evidence in intact cells for a role for Mn2+ and the spoT gene product in ppGpp degradation, and provide further evidence for an involvement of spoT and possibly divalent ions in ppGpp synthesis.     

Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations

It was known previously that 1) the relA gene of Escherichia coli encodes an enzyme capable of guanosine 3',5'-bispyrophosphate (ppGpp) synthesis, 2) an uncharacterized source of ppGpp synthesis exists in relA null strains, and 3) cellular degradation of ppGpp is mainly due to a manganese-dependent ppGpp 3'-pyrophosphohydrolase encoded by the spoT gene. Here, the effects of spoT gene insertions and deletions are compared with analogous alterations in neighboring genes in the spo operon and found to be lethal in relA+ strains as well as slower growing in relAl backgrounds than delta relA hosts. Cells with null alleles in both the relA and spoT genes are found no longer to accumulate ppGpp after glucose exhaustion or after chelation of manganese ions by picolinic acid addition; the inability to form ppGpp is reversed by a minimal spoT gene on a multicopy plasmid. Strains apparently lacking ppGpp show a complex phenotype including auxotrophy for several amino acids and morphological alterations. We propose that the SpoT protein can either catalyze or control the alternative pathway of ppGpp synthesis in addition to its known role as a (p)ppGpp 3'-pyrophosphohydrolase. We favor the possibility that the SpoT protein is a bifunctional enzyme capable of catalyzing either ppGpp synthesis or degradation.     

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.

The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.     

Synthesis of guanosine 5'-diphosphate, 3'-diphosphate in spot mutants of Escherichia coli.

The synthesis of ppGpp in spoT- mutants of Escherichia coli has been invesitgated. In these mutants the first-order rate constant for ppGpp breakdown is low, and pppGpp is barely detectable. It is shown that the rate of pppGpp, and hence ppGpp, synthesis is strongly reduced compared with that observed in spot+ strains. The low rate of magic spot synthesis satisfactorily explains the low levels of pppGpp in spoT- mutants. The pentaphosphate very probably is the precursor of ppGpp as it is in wild-type, i.e. spoT+, strains.     

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.     

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.     

Feedback control of ribosome function in Escherichia coli

We have previously proposed that the rate of ribosome function during balanced growth in E. coli, expressed as the rate of peptide chain elongation, is adjusted by a feedback mechanism: whenever that rate is submaximal (i.e. below 22 amino acid residues polymerized per active ribosome at 37 degrees C), the feedback signal ppGpp is generated by an activation of the ppGpp synthetase expressed from the spoT gene. The accumulation of ppGpp reduces the synthesis of additional ribosomes and thereby reduces the consumption of amino acids which, in turn, allows the remaining ribosomes to function at a higher rate. Here we have described with supporting evidence the proposed feedback loop in greater detail and provided a mathematical analysis which predicts that the SpoT ppGpp synthetase activity should be highest when the ribosomes function at their half-maximal rate. This prediction is consistent with reported observations and is independent of the particular (unknown) mechanism by which the rate of translation controls the ppGpp synthetase activity of SpoT.     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

Functional analysis of spoT, relA and dksA genes on quinolone tolerance in Pseudomonas aeruginosa under nongrowing condition

To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of the genes involved or linked with the stringent response, such as relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of 8 microg/ml of ofloxacin and 1 microg/ml of ciprofloxacin were shown to be approximately 20-180 and 10-40 times respectively, higher than the same for the wild type strain. The intracellular levels of ppGpp determined with high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa.     

Expression of the amino acid operons in Escherichia coli strains with an altered transcription and translation apparatus. IV. The effect of mutations disturbing the coupling of the transcription and translation processes on ilv-operon expression in cells carrying the mutation in the spoT gene

The rate of adaptation of Escherichia coli K-12 NF930 spoT1 cells with elevated intracellular level of ppGpp to various minimal media was studied. It has been found that the rate of adaptation of spoT cells, like that of parent and rel strains, depends mainly on the rate of derepression of the ilv operon. The maximal rate of the ilv operon derepression was observed when an optimal concentration of ppGpp was maintained in cells. Derepression of the ilv operon is sharply delayed when the level of ppGpp is elevated or reduced. Mutations altering the translation system do not change the rate of adaptation of spoT cells. Rifampicin resistance mutations which altered the structure of RNA polymerase change the rate of adaptation of spoT cells to minimal media, especially to those containing serine at high concentrations. The possible role of serine in the regulation of ppGpp degradation system is discussed.     

SpoT governs Legionella pneumophila differentiation in host macrophages

During its life cycle, Legionella pneumophila alternates between a replicative and a transmissive state. To determine their contributions to L. pneumophila differentiation, the two ppGpp synthetases, RelA and SpoT, were disrupted. Synthesis of ppGpp was required for transmission, as relA spoT mutants were killed during entry to and exit from macrophages. RelA, which senses amino acid starvation induced by serine hydroxamate, is dispensable in macrophages, as relA mutants spread efficiently. SpoT monitors fatty acid biosynthesis (FAB), since following cerulenin treatment, wild-type and relA strains expressed the flaA transmissive gene, but relA spoT mutants did not. As in Escherichia coli , the SpoT response to FAB perturbation likely required an interaction with acyl-carrier protein (ACP), as judged by the failure of the spoT-A413E allele to rescue transmissive trait expression of relA spoT bacteria. Furthermore, SpoT was essential for transmission between macrophages, since secondary infections by relA spoT mutants were restored by induction of spoT , but not relA . To resume replication, ppGpp must be degraded, as mutants lacking spoT hydrolase activity failed to convert from the transmissive to the replicative phase in either bacteriological medium or macrophages. Thus, L. pneumophila requires SpoT to monitor FAB and to alternate between replication and transmission in macrophages.     

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli , V. cholerae Δ relA Δ spoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae . By repeated subculturing of a V. cholerae Δ relA Δ spoT mutant, a suppressor strain with (p)ppGpp⁰ phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene ( VC1224 ), which codes for a small protein (∼29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV . Using E. coli Δ relA or Δ relA Δ spoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (Δ relA Δ spoT relV676 ), which seems to be responsible for the (p)ppGpp⁰ phenotype. Analysis of a V. cholerae Δ relA Δ spoT Δ relV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.     

Guanosine 5'-diphosphate 3'-diphosphate levels, carbon source, and ribonucleic acid synthesis in a mutant strain of Escherichia coli

We have previously described a mutant strain of Escherichia coli (2S142) which shows a specific inhibition of stable RNA synthesis at 42 degrees C. The temperature-sensitive lesion mimics a carbon source downshift (diauxie lag). We therefore measured RNA synthesis and levels of ppGpp (guanosine 5'-diphosphate 3'-diphosphate) on a number of different carbon sources. There is a 6-fold variation in ppGpp levels at 42 degrees C, depending on the carbon source present. Much of the variation in ppGpp levels at 42 degrees C can be explained by variations in the decay rate of ppGpp at 42 degrees C. The rates of ribosomal RNA and total RNA synthesis also vary with the carbon source at 42 degrees C. Linear regression analysis shows only a moderately good correlation (correlation coefficient = 0.62, P = 0.0001) between the ppGpp level at 42 degrees C and the rate of rRNA synthesis at 42 degrees C. In fact, ppGpp levels are a slightly better predictor of the rate of total RNA synthesis (correlation coefficient = 0.69, P = 0.0001) at 42 degrees C. Other variables such as rate of carbon source uptake appear to have very little, if any, relationship to the rate of rRNA synthesis on the different carbon sources. Segmented linear regression analysis indicates that ppGpp levels and rates of RNA synthesis correlate best when the carbon sources are divided into two groups: 6- and 12-carbon sugars and other carbon sources. The rate of rRNA synthesis in 2S142 at 42 degrees C appears to be relatively insensitive to ppGpp levels with 6- and 12-carbon sugars as the carbon source. These data raise the possibility that carbon source may affect rRNA synthesis in a manner that is at least partially unrelated to ppGpp levels.     

Characterization of the guanosine 5'-triphosphate 3'-diphosphate and guanosine 5'-diphosphate 3'-diphosphate degradation reaction catalyzed by a specific pyrophosphorylase from Escherichia coli.

Guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) are specifically degraded by a manganese-dependent pyrophosphorylase present in spoT+ but not in spoT- strains of Escherichia coli, indicating that the enzyme is the spoT gene product. The enzyme catalyzes the release of pyrophosphate from the 3' position of ppGpp or pppGpp, yielding ppG and pppG, respectively; pppGpp could not be detected as an intermediate in the decay reaction. Degradation of (p)ppGpp is optimal in the presence of 200 to 300 mM potassium or sodium acetate, at a pH of 7.5 to 8 and a temperature of 37 degrees C.     

Guanosine 3',5'-bispyrophosphate (ppGpp) synthesis in cells of Escherichia coli starved for Pi.

Cells of Escherichia coli which enter a phase of starvation for Pi induce the synthesis of the nucleotide guanosine 3',5'-bispyrophosphate (ppGpp). This induction is relA independent but depends on the spoT gene product. A mutant unable to produce ppGpp is impaired in the expression of two genes which belong to the pho regulon, a defect which is dependent on the product of spoT. We suggest that ppGpp is essential for the proper induction of the genes which belong to the pho regulon.