ppGpp介导的抗生素胁迫应答机制研究进展

抗生素是由微生物在生长发育后期产生的次级代谢产物,具有杀死或抑制细菌生长的能力,因此被广泛应用于细菌感染的临床治疗。在长期的进化过程中,细菌采取多种方式应对环境中抗生素的威胁。除了广为人知的抗生素耐药性(resistance)之外,细菌还能对抗生素产生耐受性(tolerance)和持留性(persistence),严重影响抗生素的临床疗效。鸟苷四磷酸(guanosine tetraphosphate, ppGpp)和鸟苷五磷酸(guanosine pentaphosphate, pppGpp) (本文统称ppGpp)是细菌应对营养饥饿等不利环境时产生的"报警"信号分子,其能够在全局水平调控基因的表达,使细菌适应不利的环境。越来越多的研究表明,ppGpp与细菌应对抗生素胁迫密切相关。基于此,本文综述了细菌中ppGpp的合成与水解及其作用机制,并重点阐述了ppGpp介导抗生素胁迫应答的分子机制,以期为新型抗生素的开发提供新思路。     

信号分子ppGpp与微生物环境适应性

微生物能感知环境胁迫信号,通过触发严谨反应对生长速率进行调节,并通过一系列代谢调控,使细胞能在不利环境中生存。高度磷酸化的鸟苷四/五磷酸ppGpp/pppGpp（文中以ppGpp统称）作为信号分子对微生物生理具有广泛的调节作用,至今仍是微生物学研究热点之一。ppGpp对于微生物适应高温、高压等环境起到了积极的作用。综述了信号分子ppGpp合成降解机制及其调控微生物适应性方面的研究进展。     

(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。     

Studies on the Degradation Mechanisms of Proteins and their Derivatives in the Foods

The volatile sulfur components produced by boiling soybean meal hydrolyzates (AMINOSAN-EKI) have been identified as dimethyl sulfide and hydrogen sulfide. No mercaptan or disulfides were detected.

The main precursor of dimethyl sulfide is supposed to be methionine methylsulfonium compound derived from methionine and pectin substances (–COOCH₃) during the hydrolysis of soybean meal by hydrochloric acid.     

Molecular dissection of the mycobacterial stringent response protein Rel

Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram-negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of Rel(Msm) is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full-length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Full-length Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full-length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA-mediated synthesis of ppGpp.     

ppGpp and RNA-polymerase backtracking guide antibiotic-induced mutable gambler cells

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The role of the omega subunit of RNA polymerase in expression of the relA gene in Escherichia coli

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level.     

The Alarmone (p)ppGpp Regulates Primer Extension by Bacterial Primase

Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis. Bacterial primase activity can be regulated by the starvation-inducible nucleotide (p)ppGpp. This regulation contributes to a timely inhibition of DNA replication upon amino acid starvation in the Gram-positive bacterium Bacillus subtilis. Here, we characterize the effect of (p)ppGpp on B. subtilis DnaG primase activity in vitro. Using a single-nucleotide resolution primase assay, we dissected the effect of ppGpp on the initiation, extension, and fidelity of B. subtilis primase. We found that ppGpp has a mild effect on initiation, but strongly inhibits primer extension and reduces primase processivity, promoting termination of primer extension. High (p)ppGpp concentration, together with low GTP concentration, additively inhibit primase activity. This explains the strong inhibition of replication elongation during starvation which induces high levels of (p)ppGpp and depletion of GTP in B. subtilis. Finally, we found that lowering GTP concentration results in mismatches in primer base pairing that allow priming readthrough, and that ppGpp reduces readthrough to protect priming fidelity. These results highlight the importance of (p)ppGpp in protecting replisome integrity and genome stability in fluctuating nucleotide concentrations upon onset of environmental stress.     

mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria

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The A-factor regulatory cascade and cAMP in the regulation of physiological and morphological development in Streptomyces griseus

In the A-factor regulatory cascade leading to the onset of streptomycin biosynthesis and aerial mycelium formation in Streptomyces griseus, the A-factor receptor protein (ArpA) serves as a DNA-binding repressor and A-factor releases the repression by binding to ArpA and dissociating it from the DNA. Mutants defective in arpA therefore produce streptomycin and aerial hyphae in the absence of A-factor. A gene that inhibits streptomycin production and aerial hyphae formation in an arpA mutant was cloned on a high-copy-number plasmid and found to encode a eukaryotic-type adenylate cyclase (CyaA). Consistent with this, an exogenous supply of cAMP at high concentration almost abolished streptomycin production and aerial hyphae formation. On the other hand, cAMP at lower concentrations stimulated or accelerated these developmental processes. The effects of cAMP were detectable only in arpA mutants, and not in the wild-type strain; an exogenous supply of cAMP or cyaA disruption in the wild-type strain caused almost no effect on these phenotypes. Thus the effects of cAMP became apparent only in the arpA-defective background. cAMP at high concentrations inhibited stringent response factor ppGpp production, which is important for the onset of antibiotic biosynthesis. cAMP also influenced the timing of tyrosine phosphorylation of more than nine proteins. These findings show that a cAMP regulatory relay for physiological and morphological development functions in a concerted and interdependent way with other signal transduction pathways. Journal of Industrial Microbiology & Biotechnology (2001) 27, 177–182. Received 21 September 1999/ Accepted in revised form 14 September 2000     

Structural basis for c-di-AMP–dependent regulation of the bacterial stringent response by receptor protein DarB

The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress conditions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3′3′-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide molecules or two AMP molecules; only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3′3′-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB–Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB–Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regulation by c-di-AMP.     

Fluorescent monitoring of cellular physiological status depending on the accumulation of ppGpp

For evaluating the physiological status of cells, astringent response network was used. Fluorescence from intact E. coli, which has a plasmid encoding the green fluorescence protein (GFP) under the regulation of rpoS promoter, was monitored. Comparison of the response of different E. coli strains demonstrated an essential role of ppGpp in the expression of GFP, as it activated the rpoS promoter. The physiological status of intact cells, that depends on ppGpp accumulation in response to the nutritional status such as amino acid starvation, could therefore be monitored by measuring fluorescent intensity using this reporter gene.     

真核生物mRNA非翻译区结构特征与mRNA翻译

真核生物中蛋白质合成通常在翻译水平受到调控,而mRNA非翻译区与mRNA翻译之间关系密切。真核生物mRNA非翻译区长度、mRNA二级结构、GC含量、顺式作用元件与反式作用因子、mRNA帽端结构和多聚腺苷酸尾等结构对翻译具有重要的调控作用。本文就真核生物mRNA非翻译区结构特征对其翻译的影响做一综述。