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Tozawa Y Nozawa A Kanno T Narisawa T Masuda S Kasai K Nanamiya H 《The Journal of biological chemistry》2007,282(49):35536-35545
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The current dogma implicating RelA as the sole enzyme controlling (p)ppGpp production and degradation in Gram-positive bacteria does not apply to Streptococcus mutans. We have now identified and characterized two genes, designated as relP and relQ, encoding novel enzymes that are directly involved in (p)ppGpp synthesis. Additionally, relP is co-transcribed with a two-component signal transduction system (TCS). Analysis of the (p)ppGpp synthetic capacity of various mutants and the behaviour of strains lacking combinations of the synthetase enzymes have revealed a complex regulon and fundamental differences in the way S. mutans manages alarmone production compared with bacterial paradigms. The functionality of the RelP and RelQ enzymes was further confirmed by demonstrating that expression of relP and relQ restored growth of a (p)ppGpp(0) Escherichia coli strain in minimal medium, SMG and on medium containing 3-amino-1,2,4-triazole, and by demonstrating (p)ppGpp production in various complemented mutant strains of E. coli and S. mutans. Notably, RelQ, and RelP and the associated TCS, are harboured in some, but not all, pathogenic streptococci and related Gram-positive organisms, opening a new avenue to explore the variety of strategies employed by human and animal pathogens to survive in adverse conditions that are peculiar to environments in their hosts. 相似文献
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Characterization of pyrimidine-repressible and arginine-repressible carbamyl phosphate synthetases from Bacillus subtilis. 总被引:1,自引:0,他引:1
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The number and properties of carbamyl phosphate synthetases in Bacillus subtilis have been uncertain because of conflicting genetic results and instability of the enzyme in extracts. The discovery of a previously unrecognized requirement of B. subtilis carbamyl phosphate synthetases for a high concentration of potassium ions for activity and stability permitted unequivocal demonstration that this bacterium elaborates two carbamyl phosphate synthetases. Carbamyl phosphate synthetase A was shown to be repressed by arginine, to have a molecular weight of about 200,000, and to be coded for by a gene that maps near argC4. This isozyme was insensitive to metabolites of the arginine and pyrimidine biosynthetic pathways. Carbamyl phosphate synthetase P was found to be repressed by uracil, to have a molecular weight of 90,000 to 100,000, and to be coded for by a gene that maps near the other pyr genes. This isozyme was activated by phosphoridine nucleotides. Other kinetic properties of the two isozymes were compared. Bacillus thus resembles eucaryotic microbes in producing two carbamyl phosphate synthetases, rather than the enteric bacteria, which produce a single carbamyl phosphate synthetase. 相似文献
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RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the "stringent response" to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome. 相似文献
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A hyperphosphorylated guanosine nucleotide, (p)ppGpp, was initially identified as the effector molecule responsible for the stringent response in Escherichia coli. However, a rapidly growing number of reports proves that (p)ppGpp-mediated regulation is conserved in many bacteria and even in plants. It is now clear that (p)ppGpp acts as a global regulator during physiological adaptation of the organism to a plethora of environmental conditions. Adaptation is not only essential for surviving periods of stress and nutrient exhaustion but also for the interaction of bacteria with their eukaryotic host, as observed during pathogenesis and symbiosis, and for bacterial multicellular behaviour. Recently, there have been several new discoveries about the effects of (p)ppGpp levels, balanced by RelA-SpoT homologue proteins, in diverse organisms. 相似文献
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Enzymes of the Rel/Spo family enable bacteria to survive prolonged periods of nutrient limitation by producing an intracellular signaling alarmone, (p)ppGpp, which triggers the so-called stringent response. Both the synthesis of (p)ppGpp from ATP and GDP(GTP), and its hydrolysis to GDP(GTP) and pyrophosphate, are catalyzed by Rel/Spo proteins. The 2.1 A crystal structure of the bifunctional catalytic fragment of the Rel/Spo homolog from Streptococcus dysgalactiae subsp. equisimilis, Rel(Seq), reveals two conformations of the enzyme corresponding to known reciprocal activity states: (p)ppGpp-hydrolase-OFF/(p)ppGpp-synthetase-ON and hydrolase-ON/synthetase-OFF. The hydrolase and synthetase domains bear remarkable similarities to the catalytic domains of the cyclic phosphodiesterase and nucleotidyltransferase superfamilies, respectively. The active sites, separated by more than 30 A, contain bound nucleotides including an unusual (p)ppGpp derivative, GDP-2':3'-cyclic monophosphate. Reciprocal regulation of the antagonistic catalytic activities, suggested by the structure, is supported by mutagenesis experiments and appears to involve ligand-induced signal transmission between the two active sites. 相似文献
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Bacteria inhabit diverse environmental niches and consequently must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as maintaining cellular homeostasis and regulating bacterial interactions with a host, other bacteria, or phages. (p)ppGpp produced by pathogenic bacteria facilitates the evasion of host defenses such as reactive nitrogen intermediates, acidic pH, and the complement system. Additionally, (p)ppGpp and pyrophosphorylated derivatives of canonical adenosine nucleotides called (p)ppApp are emerging as effectors of bacterial toxin proteins. Here, we review the RSH protein family with a focus on its unconventional roles during host infection and bacterial competition. 相似文献
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The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases. 相似文献
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By use of degenerate primers, we amplified a fragment of a relA/spoT homologous gene from Bacillus stearothermophilus. Chromosomal walking enabled us to sequence the entire gene and its flanking regions. The primary sequence of the gene product is 78% identical to the RelA/SpoT homologue of Bacillus subtilis and both gene loci share a similar genetic organization. The B. stearothermophilus rel gene was analyzed in vivo by heterologous expression in the B. subtilis relA deletion strain TW30, and is shown to complement the growth defects of TW30. The recombinant RelBst protein was detected by Western immunoanalysis, and synthesizes guanosine-3'-diphosphate-5'-(tri)diphosphate ((p)ppGpp) after amino acid stress and carbon starvation. These in vivo data, the genetic organization, and the primary structure compared to other RelA/SpoT homologues provide circumstantial evidence that the identified gene encodes the only (p)ppGpp synthetase in B. stearothermophilus presumed to serve also as (p)ppGpp hydrolase. 相似文献
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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. 相似文献
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Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation. 相似文献
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Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism 总被引:1,自引:0,他引:1
Bacteria respond to nutritional stresses by producing an intracellular alarmone, guanosine 5'-(tri)diphosphate, 3'-diphosphate [(p)ppGpp], which triggers the stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, upon fatty acid or carbon starvation, SpoT enzyme activity switches from (p)ppGpp degradation to (p)ppGpp synthesis, but the signal and mechanism for this response remain totally unknown. Here, we characterize for the first time a physical interaction between SpoT and acyl carrier protein (ACP) using affinity co-purifications and two-hybrid in E. coli. ACP, as a central cofactor in fatty acid synthesis, may be an ideal candidate as a mediator signalling starvation to SpoT. Accordingly, we show that the ACP/SpoT interaction is specific of SpoT and ACP functions because ACP does not interact with the homologous RelA protein and because SpoT does not interact with a non-functional ACP. Using truncated SpoT fusion proteins, we demonstrate further that ACP binds the central TGS domain of SpoT, consistent with a role in regulation. The behaviours of SpoT point mutants that do not interact with ACP reveal modifications of the balance between the two opposite SpoT catalytic activities thereby changing (p)ppGpp levels. More importantly, these mutants fail to trigger (p)ppGpp accumulation in response to fatty acid synthesis inhibition, supporting the hypothesis that the ACP/SpoT interaction may be involved in SpoT-dependent stress response. This leads us to propose a model in which ACP carries information describing the status of cellular fatty acid metabolism, which in turn can trigger the conformational switch in SpoT leading to (p)ppGpp accumulation. 相似文献
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Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genes dhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed. 相似文献
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Hanwen Li Jinqiang Nian Shuang Fang Meng Guo Xiahe Huang Fengxia Zhang Qing Wang Jian Zhang Jiaoteng Bai Guojun Dong Peiyong Xin Xianzhi Xie Fan Chen Guodong Wang Yingchun Wang Qian Qian Jianru Zuo Jinfang Chu Xiaohui Ma 《遗传学报》2022,49(5):469-480
Nitrogen is an essential macronutrient for all living organisms and is critical for crop productivity and quality.In higher plants, inorganic nitrogen is absorbed through roots and then assimilated into amino acids by the highly conserved glutamine synthetase/glutamine:2-oxoglutarate aminotransferase(GS/GOGAT) cycle.How nitrogen metabolism and nitrogen starvation responses of plants are regulated remains largely unknown. Previous studies revealed that mutations in the rice ABNORMAL CYTOKININ RES... 相似文献
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Independent genes for two threonyl-tRNA synthetases in Bacillus subtilis. 总被引:11,自引:4,他引:7
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With the exception of Escherichia coli lysyl-tRNA synthetase, the genes coding for the different aminoacyl-tRNA synthetases in procaryotes are always unique. Here we report on the occurrence and cloning of two genes (thrSv and thrS2), both encoding functional threonyl-tRNA synthetase in Bacillus subtilis. The two proteins share only 51.5% identical residues, which makes them almost as distinct from each other as each is from E. coli threonyl-tRNA synthetase (42 and 47%). Both proteins complement an E. coli thrS mutant and effectively charge E. coli threonyl tRNA in vitro. Their genes have been mapped to 250 degrees (thrSv) and 344 degrees (thrS2) on the B. subtilis chromosome. The regulatory regions of both genes are quite complex and show structural similarities. During vegetative growth, only the thrSv gene is expressed. 相似文献
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Stringent response of Bacillus stearothermophilus: evidence for the existence of two distinct guanosine 3',5'-polyphosphate synthetases. 总被引:6,自引:4,他引:2
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Bacillus stearothermophilus reacted to pseudomonic acid-induced inhibition of isoleucine-transfer ribonucleic acid (RNA) acylation and to energy downshift caused by alpha-methylglucoside addition with accumulation of guanosine 3',5'-polyphosphates [(p)ppGpp] and restriction of RNA synthesis. In vitro studies indicated that (p)ppGpp was synthesized by two different enzymes. One enzyme, (p)ppGpp synthetase I, was present in the ribosomal fraction, required the addition of a ribosome-messenger RNA-transfer RNA complex for activation, and was inhibited by tetracycline and thiostrepton. It is suggested that (p)ppGpp synthetase I is comparable to the relA gene product from Escherichia coli and is responsible for (p)ppGpp accumulation during amino acid starvation. The other enzyme, (p)ppGpp synthetase II, was found in the high-speed supernatant fraction (S100). It functioned independently of ribosomes, transfer RNA, and messenger RNA and was not inhibited by the above-mentioned antibiotics. (p)ppGpp synthetase II is thought to be responsible for (p)ppGpp accumulation during carbon source downshift. The two enzymes differ in their Km values for adenosine triphosphate (ATP):2mM ATP for synthetase I and 0.05 mM ATP for synthetase II. They also have different molecular weights: apparent Mr of 86,000 (+/- 5,000) for synthetase I and 74,000 (+/- 5,000) for synthetase II. 相似文献