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。     

Regulation of cell growth during serum starvation and bacterial survival in macrophages by the bifunctional enzyme SpoT in Helicobacter pylori

In Helicobacter pylori the stringent response is mediated solely by spoT. The spoT gene is known to encode (p)ppGpp synthetase activity and is required for H. pylori survival in the stationary phase. However, neither the hydrolase activity of the H. pylori SpoT protein nor the role of SpoT in the regulation of growth during serum starvation and intracellular survival of H. pylori in macrophages has been determined. In this study, we examined the effects of SpoT on these factors. Our results showed that the H. pylori spoT gene encodes a bifunctional enzyme with both a hydrolase activity and the previously described (p)ppGpp synthetase activity, as determined by introducing the gene into Escherichia coli relA and spoT defective strains. Also, we found that SpoT mediates a serum starvation response, which not only restricts the growth but also maintains the helical morphology of H. pylori. Strikingly, a spoT null mutant was able to grow to a higher density in serum-free medium than the wild-type strain, mimicking the “relaxed” growth phenotype of an E. coli relA mutant during amino acid starvation. Finally, SpoT was found to be important for intracellular survival in macrophages during phagocytosis. The unique role of (p)ppGpp in cell growth during serum starvation, in the stress response, and in the persistence of H. pylori is discussed.     

Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.     

假单胞菌M18 relA 突变株的构建及其对吩嗪-1-羧酸合成的调控

假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素两种抗生素的植物根际分离细菌。RelA催化合成的效应分子ppGpp能介导细菌因营养饥饿引起的应激反应。以M18菌株染色体DNA为模板,PCR扩增获得relA基因,通过庆大霉素抗性片段插入失活与同源重组技术,构建假单胞菌M18的relA突变菌株M18RAG。在PPM培养基中进行PCA发酵分析,发现突变菌株M18RAG的PCA产量显著升高,约为野生型菌株的1.5-2倍。relA基因反式互补实验以及phzA′-′lacZ翻译融合测定结果,均进一步证明了RelA对PCA生物合成及其基因表达具有抑制作用。     

Cholera Toxin Production during Anaerobic Trimethylamine N-Oxide Respiration Is Mediated by Stringent Response in Vibrio cholerae

As a facultative anaerobe, Vibrio cholerae can grow by anaerobic respiration. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly promoted during anaerobic growth using trimethylamine N-oxide (TMAO) as an alternative electron acceptor. Here, we investigated the molecular mechanisms of TMAO-stimulated CT production and uncovered the crucial involvement of stringent response in this process. V. cholerae 7th pandemic strain N16961 produced a significantly elevated level of ppGpp, the bacterial stringent response alarmone, during anaerobic TMAO respiration. Bacterial viability was impaired, and DNA replication was also affected under the same growth condition, further suggesting that stringent response is induced. A ΔrelA ΔspoT ppGpp overproducer strain produced an enhanced level of CT, whereas anaerobic growth via TMAO respiration was severely inhibited. In contrast, a ppGpp-null strain (ΔrelA ΔspoT ΔrelV) grew substantially better, but produced no CT, suggesting that CT production and bacterial growth are inversely regulated in response to ppGpp accumulation. Bacterial capability to produce CT was completely lost when the dksA gene, which encodes a protein that works cooperatively with ppGpp, was deleted. In the ΔdksA mutant, stringent response growth inhibition was alleviated, further supporting the inverse regulation of CT production and anaerobic growth. In vivo virulence of ΔrelA ΔspoT ΔrelV or ΔdksA mutants was significantly attenuated. The ΔrelA ΔspoT mutant maintained virulence when infected with exogenous TMAO despite its defective growth. Together, our results reveal that stringent response is activated under TMAO-stimulated anaerobic growth, and it regulates CT production in a growth-dependent manner in V. cholerae.     

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.     

Accumulation of guanosine tetraphosphate induced by polymixin and gramicidin in Escherichia coli

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA⁺) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.     

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.     

The regulation of the ammonia assimilatory enzymes in Rel+ and Rel- strains of Salmonella typhimurium

Summary The influence of the relA1 mutation on the regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 1.4.1.3), was examined. When cells grown in rich media (either Luria broth or glucose-ammonia plus casamino acids) were transferred to a glucose-ammonia medium, the relA mutant failed to resume growth and did not have the same increase in any of the assimilatory enzyme activities as the rel ⁺ strain. This effect was particularly dramatic for glutamate dehydrogenase, which increased 6-fold in the rel ⁺ strain. Measurements of the guanosine nucleotide concentrations showed that the rel ⁺ strain had a ppGpp concentration about 9 times that of the relA mutant 5 min after the shift to minimal medium. These results are consistent with those for other biosynthetic enzymes and show that the ammonia assimilatory enzymes require a relA product for their synthesis during shifts from rich to minimal media. In addition, we examined the response of these strains to a change in nitrogen source. The relA mutant again failed to resume growth after a shift from glucose-ammonia to glucose-arginine medium. Even though the ppGpp concentration did not increase, the rel ⁺ strain grew and increased glutamine synthetase activities about 2-fold. These changes in the absence of increased ppGpp levels suggest that some other relA-mediated function is important during this change in nitrogen source.     

A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro

A gene encoding a putative guanosine 3′,5′-bispyrophosphate (ppGpp) synthase–degradase, designated Cr-RSH, was identified in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii. The encoded Cr-RSH protein possesses a putative chloroplast-targeting signal at its NH₂-terminus, and translocation of Cr-RSH into chloroplasts isolated from C.reinhardtii was demonstrated in vitro. The predicted mature region of Cr-RSH exhibits marked similarity to eubacterial members of the RelA–SpoT family of proteins. Expression of an NH₂-terminal portion of Cr-RSH containing the putative ppGpp synthase domain in a relA, spoT double mutant of Escherichia coli complemented the growth deficits of the mutant cells. Chromatographic analysis of ³²P-labeled cellular mononucleotides also revealed that expression of Cr-RSH in the mutant bacterial cells resulted in the synthesis of ppGpp. SpoT, which catalyzes (p)ppGpp degradation, is dispensable in E.coli only if cells also lack RelA, which possesses (p)ppGpp synthase activity. The complementation analysis thus indicated that Cr-RSH possesses both ppGpp synthase and degradase activities. These results represent the first demonstration of ppGpp synthase–degradase activities in a eukaryotic organism, and they suggest that eubacterial stringent control mediated by ppGpp has been conserved during evolution of the chloroplast from a photosynthetic bacterial symbiont.     

Systematic Production of Inactivating and Non-Inactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli

In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into relA⁺ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase.     

SpoT-triggered stringent response controls usp gene expression in Pseudomonas aeruginosa

The universal stress proteins (Usps) UspK (PA3309) and UspN (PA4352) of Pseudomonas aeruginosa are essential for surviving specific anaerobic energy stress conditions such as pyruvate fermentation and anaerobic stationary phase. Expression of the respective genes is under the control of the oxygen-sensing regulator Anr. In this study we investigated the regulation of uspN and three additional P. aeruginosa usp genes: uspL (PA1789), uspM (PA4328), and uspO (PA5027). Anr induces expression of these genes in response to anaerobic conditions. Using promoter-lacZ fusions, we showed that P_uspL-lacZ, P_uspM-lacZ, and P_uspO-lacZ were also induced in stationary phase as described for P_uspN-lacZ. However, stationary phase gene expression was abolished in the P. aeruginosa triple mutant Δanr ΔrelA ΔspoT. The relA and spoT genes encode the regulatory components of the stringent response. We determined pppGpp and ppGpp levels using a thin-layer chromatography approach and detected the accumulation of ppGpp in the wild type and the ΔrelA mutant in stationary phase, indicating a SpoT-derived control of ppGpp accumulation. Additional investigation of stationary phase in LB medium revealed that alkaline pH values are involved in the regulatory process of ppGpp accumulation.     

The Role of relA and spoT in Yersinia pestis KIM5+ Pathogenicity

The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a ΔrelA ΔspoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26°C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was>1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5×10⁴ CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×10⁵ CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×10⁴ CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague.