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 ...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 RESPONSE1(ABC1)gene encoding Fd-GOGAT cause a typical nitrogen deficiency syndrome.Here,we show that ARE2(for ABC1 REPRESSOR2)is a key regulator of nitrogen starvation responses in rice.The are2 mutations partially rescue the nitrogen-deficient phenotype of abc1 and the are2 mutants show enhanced tolerance to nitrogen deficiency,suggesting that ARE2 genetically interacts with ABC1/Fd-GOGAT.ARE2 encodes a chloroplast-localized Rel A/Spo T homolog protein that catalyzes the hydrolysis of guanosine pentaphosphate or tetraphosphate(p)pp Gpp,an alarmone regulating the stringent response in bacteria under nutritional stress conditions.The are2 mutants accumulate excessive amounts of(p)pp Gpp,which correlate with lower levels of photosynthetic proteins and higher amino acid levels.Collectively,these observations suggest that the alarmone(p)pp Gpp mediates nitrogen stress responses and may constitute a highly conserved mechanism from bacteria to plants.展开更多
More modifiable sites on the nucleoside motif may need to be explored for developing novel(p)ppGpp molecular tools.Herein,we report for the first time that the C7-substituted deazapurine nucleoside triphosphates beari...More modifiable sites on the nucleoside motif may need to be explored for developing novel(p)ppGpp molecular tools.Herein,we report for the first time that the C7-substituted deazapurine nucleoside triphosphates bearing small modifications as substrates could be effectively accepted by RelSeqNTD protein to react with ATP to give pppGpp derivatives with 65%—89%yields.Further structural derivatization via metal-coupling reaction was performed to produce C7-substituted GTP derivatives with larger bulkiness,and those GTP derivatives were also proven to be good substrates of RelSeqNTD protein.Alkynyl modified pppGpp could be coupled with probes by click reactions as the potential molecular tools for fishing proteins in biological research.We further explored whether the C7-alkynyl-pppGpp(pppGEpp)could be recognized by pppGpp interaction proteins.A micromolar level binding affinity(with a KD value of less than 10μM)between pppGpp(pppGEpp)and its binding proteins was obtained from the Isothermal Titration Curve(ITC).All those illustrate that these easily accessible functionalized C7-substituted pppGpp derivatives were suitable tools for further exploring the molecular interaction between pppGpp and its binding proteins.展开更多
Antibiotics combat bacteria through their bacteriostatic(by growth inhibition)or bactericidal(by killing bacteria)action.Mechanistically,it has been proposed that bactericidal antibiotics trigger cellular damage,while...Antibiotics combat bacteria through their bacteriostatic(by growth inhibition)or bactericidal(by killing bacteria)action.Mechanistically,it has been proposed that bactericidal antibiotics trigger cellular damage,while bacteriostatic antibiotics suppress cellular metabolism.Here,we demonstrate how the difference between bacteriostatic and bactericidal activities of the antibiotic chloramphenicol can be attributed to an antibiotic-induced bacterial protective response:the stringent response.Chloramphenicol targets the ribosome to inhibit the growth of the Gram-positive bacterium Bacillus subtilis.Intriguingly,we found that chloramphenicol becomes bactericidal in B.subtilis mutants unable to produce(p)ppGpp.We observed a similar(p)ppGpp-dependent bactericidal effect of chloramphenicol in the Gram-positive pathogen Enterococcus faecalis.In B.subtilis,chloramphenicol treatment induces(p)ppGpp accumulation through the action of the(p)ppGpp synthetase RelA.(p)ppGpp subsequently depletes the intracellular concentration of GTP and antagonizes GTP action.This GTP regulation is critical for preventing chloramphenicol from killing B.subtilis,as bypassing(p)ppGpp-dependent GTP regulation potentiates chloramphenicol killing,while reducing GTP synthesis increases survival.Finally,chloramphenicol treatment protects cells from the classical bactericidal antibiotic vancomycin,reminiscent of the clinical phenomenon of antibiotic antagonism.Taken together,our findings suggest a role of(p)ppGpp in the control of the bacteriostatic and bactericidal activity of antibiotics in Gram-positive bacteria,which can be exploited to potentiate the efficacy of existing antibiotics.展开更多
Sleep is essential for animals,and receives inputs from circadian,homeostasis,and environment,yet the mechanisms of sleep regulation remain elusive.Discovery of molecules in living systems and demonstration of their f...Sleep is essential for animals,and receives inputs from circadian,homeostasis,and environment,yet the mechanisms of sleep regulation remain elusive.Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology.Here,we report that guanosine-50-diphos-phate,30-diphosphate(ppGpp)is present in Drosophila,and plays an important role in regulation of sleep and starvation-induced sleep loss(SISL).ppGpp is detected in germ-free Drosophila and hydrolyzed by an enzyme encoded by the mesh1 gene in Drosophila.Nighttime sleep and SISL were defected in mesh1 mutant flies,and rescued by expression of wildtype Mesh1,but not the enzymatically defective mutant Mesh1E66A.Ectopic expression of RelA,the Escherichia coli synthetase for ppGpp,phenocopied mesh1 knockout mutants,whereas overexpression of Mesh1 resulted in the opposite phenotypes,supporting that ppGpp is both necessary and sufficient in sleep regulation.A chemo connectomic screen followed by genetic intersection experiments implicates the Dilp2 neurons in the pars intercerebralis(PI)brain region as the site of ppGpp function.Our results have thus validated the presence of ppGpp in Drosophila and revealed a physiological role of ppGpp in sleep regulation for the first time.展开更多
基金supported by grants from the Ministry of Agriculture and Rural Affairs of China(2016ZX08009003-0022016ZX08009003-005 and 2016ZX08009003-004)+2 种基金Chinese Academy of Sciences(XDA08010401-2)the Ministry of Science and Technology of the People’s Republic of China(2016YFD0100706)the State Key Laboratory of Plant Genomics。
文摘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 RESPONSE1(ABC1)gene encoding Fd-GOGAT cause a typical nitrogen deficiency syndrome.Here,we show that ARE2(for ABC1 REPRESSOR2)is a key regulator of nitrogen starvation responses in rice.The are2 mutations partially rescue the nitrogen-deficient phenotype of abc1 and the are2 mutants show enhanced tolerance to nitrogen deficiency,suggesting that ARE2 genetically interacts with ABC1/Fd-GOGAT.ARE2 encodes a chloroplast-localized Rel A/Spo T homolog protein that catalyzes the hydrolysis of guanosine pentaphosphate or tetraphosphate(p)pp Gpp,an alarmone regulating the stringent response in bacteria under nutritional stress conditions.The are2 mutants accumulate excessive amounts of(p)pp Gpp,which correlate with lower levels of photosynthetic proteins and higher amino acid levels.Collectively,these observations suggest that the alarmone(p)pp Gpp mediates nitrogen stress responses and may constitute a highly conserved mechanism from bacteria to plants.
基金supported by the National Natural ScienceFoundation of China(U23A20106,22111530224,22304044 and 22473040)the Outstanding Youth Innovation Research Group in Henan Province(242300421005)Doctoral Initiation Fund(202103084).
文摘More modifiable sites on the nucleoside motif may need to be explored for developing novel(p)ppGpp molecular tools.Herein,we report for the first time that the C7-substituted deazapurine nucleoside triphosphates bearing small modifications as substrates could be effectively accepted by RelSeqNTD protein to react with ATP to give pppGpp derivatives with 65%—89%yields.Further structural derivatization via metal-coupling reaction was performed to produce C7-substituted GTP derivatives with larger bulkiness,and those GTP derivatives were also proven to be good substrates of RelSeqNTD protein.Alkynyl modified pppGpp could be coupled with probes by click reactions as the potential molecular tools for fishing proteins in biological research.We further explored whether the C7-alkynyl-pppGpp(pppGEpp)could be recognized by pppGpp interaction proteins.A micromolar level binding affinity(with a KD value of less than 10μM)between pppGpp(pppGEpp)and its binding proteins was obtained from the Isothermal Titration Curve(ITC).All those illustrate that these easily accessible functionalized C7-substituted pppGpp derivatives were suitable tools for further exploring the molecular interaction between pppGpp and its binding proteins.
基金supported,in part,by an R35 GM127088 Grant from NIGMS and a USDA Hatch Formula Grant from Wisconsin Agricultural Experiment Station WIS01740(to Jue D.Wang).
文摘Antibiotics combat bacteria through their bacteriostatic(by growth inhibition)or bactericidal(by killing bacteria)action.Mechanistically,it has been proposed that bactericidal antibiotics trigger cellular damage,while bacteriostatic antibiotics suppress cellular metabolism.Here,we demonstrate how the difference between bacteriostatic and bactericidal activities of the antibiotic chloramphenicol can be attributed to an antibiotic-induced bacterial protective response:the stringent response.Chloramphenicol targets the ribosome to inhibit the growth of the Gram-positive bacterium Bacillus subtilis.Intriguingly,we found that chloramphenicol becomes bactericidal in B.subtilis mutants unable to produce(p)ppGpp.We observed a similar(p)ppGpp-dependent bactericidal effect of chloramphenicol in the Gram-positive pathogen Enterococcus faecalis.In B.subtilis,chloramphenicol treatment induces(p)ppGpp accumulation through the action of the(p)ppGpp synthetase RelA.(p)ppGpp subsequently depletes the intracellular concentration of GTP and antagonizes GTP action.This GTP regulation is critical for preventing chloramphenicol from killing B.subtilis,as bypassing(p)ppGpp-dependent GTP regulation potentiates chloramphenicol killing,while reducing GTP synthesis increases survival.Finally,chloramphenicol treatment protects cells from the classical bactericidal antibiotic vancomycin,reminiscent of the clinical phenomenon of antibiotic antagonism.Taken together,our findings suggest a role of(p)ppGpp in the control of the bacteriostatic and bactericidal activity of antibiotics in Gram-positive bacteria,which can be exploited to potentiate the efficacy of existing antibiotics.
基金National Natural Science Foundation of China (32061143017 to Y.R.)the Research Unit of Medical Neurobi-ology,Chinese Academy of Medical Sciences (No.2019RU003)for grant support.
文摘Sleep is essential for animals,and receives inputs from circadian,homeostasis,and environment,yet the mechanisms of sleep regulation remain elusive.Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology.Here,we report that guanosine-50-diphos-phate,30-diphosphate(ppGpp)is present in Drosophila,and plays an important role in regulation of sleep and starvation-induced sleep loss(SISL).ppGpp is detected in germ-free Drosophila and hydrolyzed by an enzyme encoded by the mesh1 gene in Drosophila.Nighttime sleep and SISL were defected in mesh1 mutant flies,and rescued by expression of wildtype Mesh1,but not the enzymatically defective mutant Mesh1E66A.Ectopic expression of RelA,the Escherichia coli synthetase for ppGpp,phenocopied mesh1 knockout mutants,whereas overexpression of Mesh1 resulted in the opposite phenotypes,supporting that ppGpp is both necessary and sufficient in sleep regulation.A chemo connectomic screen followed by genetic intersection experiments implicates the Dilp2 neurons in the pars intercerebralis(PI)brain region as the site of ppGpp function.Our results have thus validated the presence of ppGpp in Drosophila and revealed a physiological role of ppGpp in sleep regulation for the first time.
