Pseudomonas aeruginosa is a ubiquitous and metabolically versatile microorganism naturally found in soil and water.It is also an opportunistic pathogen in plants,insects,animals,and humans.In response to increasing ce...Pseudomonas aeruginosa is a ubiquitous and metabolically versatile microorganism naturally found in soil and water.It is also an opportunistic pathogen in plants,insects,animals,and humans.In response to increasing cell density,P.aeruginosa uses two acylhomoserine lactone(AHL)quorum-sensing(QS)signals(i.e.,N-3-oxo-dodecanoyl homoserine lactone[3-oxo-C12-HSL]and Nbutanoyl-homoserine lactone[C4-HsL]),which regulate the expression of hundreds of genes.However,how the biosynthesis of these two QS signals is coordinated remains unknown.We studied the regulation of these two QS signals in the rhizosphere strain PA1201.PA1201 sequentially produced 3-oxo-C12-HSL and C4-HSL at the early and late growth stages,respectively.The highest 3-oxo-C12-HSL-dependent elastase activity was observed at the early stage,while the highest C4-HSL-dependent rhamnolipid production was observed at the late stage.The atypical regulator RsaL played a pivotal role in coordinating 3-oxo-C12-HSL and C4-HSL biosynthesis and QS-associated virulence.RsaL repressed las/transcription by binding the-10 and-35 boxes of the lasl promoter.In contrast,RsaL activated rhll transcription by binding the region encoding the 5'-untranslated region of the rhll mRNA.Further,RsaL repressed its own expression by binding a nucleotide motif located in the-35 box of the rsaL promoter.Thus,RsaL acts as a molecular switch that coordinates the sequential biosynthesis of AHL QS signals and differential virulence in PA1201.Finally,C4-HSL activation by RsaL was independent of the Las and Pseudomonas quinolone signal(PQS)QS signaling systems.Therefore,we propose a new model of the QS regulatory network in PA1201,in which RsaL represents a superior player acting at the top of the hierarchy.展开更多
Biocontrol strain Pseudomonas PA1201 produces pyoluteorin(Plt),which is an antimicrobial secondary metabolite.Plt represents a promising candidate pesticide due to its broad-spectrum antifungal and antibacterial activ...Biocontrol strain Pseudomonas PA1201 produces pyoluteorin(Plt),which is an antimicrobial secondary metabolite.Plt represents a promising candidate pesticide due to its broad-spectrum antifungal and antibacterial activity.Although PA1201 contains a complete genetic cluster for Plt biosynthesis,it fails to produce detectable level of Plt when grown in media typically used for Pseudomonas strains.In this study,minimum medium(MM)was found to favor Plt biosynthesis.Using the medium M,which contains all the salts of MM medium except for mannitol,as a basal medium,we compared 10 carbon sources for their ability to promote Plt biosynthesis.Fructose,mannitol,and glycerol promoted Plt biosynthesis,with fructose being the most effective carbon source.Glucose or succinic acid had no significant effect on Plt biosynthesis,but effectively antagonized fructose-dependent synthesis of Plt.Promoter-lacZ fusion reporter strains demonstrated that fructose acted through activation of the pltLABCDEFG(pltL)operon but had no effect on other genes of plt gene cluster;glucose or succinic acid antagonized fructose-dependent pltL induction.Mechanistically,fructose-mediated Plt synthesis involved carbon catabolism repression.The two-component system CbrA/CbrB and small RNA catabolite repression control Z(crcZ)were essential for fructose-induced Plt synthesis.The small RNA binding protein Hfq and Crc negatively regulated fructose-induced Plt.Taken together,this study provides a new model of fructose-dependent Plt production in PA1201 that can help improve Plt yield by biosynthetic approaches.展开更多
Histone-like nucleoid-structuring(H-NS)proteins are key regulators in gene expression silencing and in nucleoid compaction.The H-NS family member proteins MvaU in Pseudomonas aeruginosa are thought to bind the same AT...Histone-like nucleoid-structuring(H-NS)proteins are key regulators in gene expression silencing and in nucleoid compaction.The H-NS family member proteins MvaU in Pseudomonas aeruginosa are thought to bind the same AT-rich regions of chromosomes and function to coordinate the control of a common set of genes.Here,we explored the molecular mechanism by which MvaU controls PCA biosynthesis in P.aeruginosa PA1201.We present evidence suggesting that MvaU is self-regulated.Deletion of mvaU significantly increased PCA production,and PCA production sharply decreased when mvaU was over-expressed.MvaU transcriptionally repressed phz2 cluster expression and consequently reduced PCA biosynthesis.β-galactosidase assays confirmed that base pairing near the35 box is required when MvaU regulates PCA production in PA1201.Electrophoretic mobility shift assays(EMSA)and additional point mutation analysis demonstrated that MvaU directly bound to an AT-rich motif within the promoter of the phz2 cluster.Chromatin immunoprecipitation(ChIP)analysis also indicated that MvaU directly bound to the P5 region of the phz2 cluster promoter.MvaU repression of PCA biosynthesis was independent of QscR and OxyR in PA1201 and neither PCA or H2O2 were the environmental signals that induced mvaU expression.These findings detail a new MvaU-dependent regulatory pathway of PCA biosynthesis in PA1201 and provide a foundation to increase PCA fermentation titer by genetic engineering.展开更多
Some veterinary drug residues in food products and environment have been widely regarded as severe threats to human health.Rapid and simultaneous detection methods are crucial to monitor and control veterinary drug us...Some veterinary drug residues in food products and environment have been widely regarded as severe threats to human health.Rapid and simultaneous detection methods are crucial to monitor and control veterinary drug usage.Here,we propose a fluorescence biosensor utilizing immunomagnetic beads(IMBs)and quantum dots(QDs)for the rapid and simultaneous detection of 1-adamantylamine(ADA),enrofloxacin(ENR)and tilmicosin(TIL)in raw chicken meat.A pretreatment method using sodium phosphotungstate–magnesium as extraction reagent was developed to simultaneously extract ADA,ENR and TIL from chicken meat with minor interference in background or response.By adding the IMBs modified with three types of antibodies and the QD-antigens modified with three types of BSA-antigens to sample,IMBs competitively conjugated to target antigens in a sample or QD-antigens.After magnetic separation,the residual QD-antigens were adopted to collect signals using fluorescence spectroscopy.Using QDs with well separated emission peaks,the detection of one type of targets was minorly interfered by the others.Under the optimum conditions,the biosensor exhibited the limit of detection of 0.96,3.32,and 3.17 ng/mL for ADA,ENR and TIL in chicken samples,respectively,as well as good specificity.Due to the way of direct collection of signals in extracts,the tedious and complicated multiple magnetic separation and signal amplification procedures in conventional methods were avoided,thus the procedures were significantly simplified,and the reduction of the operation time of 30 min for sample pretreatment and 40 min for detection part was achieved.The biosensor might be promising in the rapid,in-field and sensitive screening of multiple veterinary drugs to ensure agriculture and food safety.展开更多
基金financially supported by grants from the National Key R&D Program of China(2018YFA0901900 to H.Y.W.)the National Natural Science Foundation of China(No.31972231 to H.Y.W)。
文摘Pseudomonas aeruginosa is a ubiquitous and metabolically versatile microorganism naturally found in soil and water.It is also an opportunistic pathogen in plants,insects,animals,and humans.In response to increasing cell density,P.aeruginosa uses two acylhomoserine lactone(AHL)quorum-sensing(QS)signals(i.e.,N-3-oxo-dodecanoyl homoserine lactone[3-oxo-C12-HSL]and Nbutanoyl-homoserine lactone[C4-HsL]),which regulate the expression of hundreds of genes.However,how the biosynthesis of these two QS signals is coordinated remains unknown.We studied the regulation of these two QS signals in the rhizosphere strain PA1201.PA1201 sequentially produced 3-oxo-C12-HSL and C4-HSL at the early and late growth stages,respectively.The highest 3-oxo-C12-HSL-dependent elastase activity was observed at the early stage,while the highest C4-HSL-dependent rhamnolipid production was observed at the late stage.The atypical regulator RsaL played a pivotal role in coordinating 3-oxo-C12-HSL and C4-HSL biosynthesis and QS-associated virulence.RsaL repressed las/transcription by binding the-10 and-35 boxes of the lasl promoter.In contrast,RsaL activated rhll transcription by binding the region encoding the 5'-untranslated region of the rhll mRNA.Further,RsaL repressed its own expression by binding a nucleotide motif located in the-35 box of the rsaL promoter.Thus,RsaL acts as a molecular switch that coordinates the sequential biosynthesis of AHL QS signals and differential virulence in PA1201.Finally,C4-HSL activation by RsaL was independent of the Las and Pseudomonas quinolone signal(PQS)QS signaling systems.Therefore,we propose a new model of the QS regulatory network in PA1201,in which RsaL represents a superior player acting at the top of the hierarchy.
文摘Biocontrol strain Pseudomonas PA1201 produces pyoluteorin(Plt),which is an antimicrobial secondary metabolite.Plt represents a promising candidate pesticide due to its broad-spectrum antifungal and antibacterial activity.Although PA1201 contains a complete genetic cluster for Plt biosynthesis,it fails to produce detectable level of Plt when grown in media typically used for Pseudomonas strains.In this study,minimum medium(MM)was found to favor Plt biosynthesis.Using the medium M,which contains all the salts of MM medium except for mannitol,as a basal medium,we compared 10 carbon sources for their ability to promote Plt biosynthesis.Fructose,mannitol,and glycerol promoted Plt biosynthesis,with fructose being the most effective carbon source.Glucose or succinic acid had no significant effect on Plt biosynthesis,but effectively antagonized fructose-dependent synthesis of Plt.Promoter-lacZ fusion reporter strains demonstrated that fructose acted through activation of the pltLABCDEFG(pltL)operon but had no effect on other genes of plt gene cluster;glucose or succinic acid antagonized fructose-dependent pltL induction.Mechanistically,fructose-mediated Plt synthesis involved carbon catabolism repression.The two-component system CbrA/CbrB and small RNA catabolite repression control Z(crcZ)were essential for fructose-induced Plt synthesis.The small RNA binding protein Hfq and Crc negatively regulated fructose-induced Plt.Taken together,this study provides a new model of fructose-dependent Plt production in PA1201 that can help improve Plt yield by biosynthetic approaches.
基金This work was financially supported by grants from National Key R&D Program of China(2018YFA0901901 to Y.-W.He)National Natural Science Foundation of China(No.31972231 to Y.-W.He).
文摘Histone-like nucleoid-structuring(H-NS)proteins are key regulators in gene expression silencing and in nucleoid compaction.The H-NS family member proteins MvaU in Pseudomonas aeruginosa are thought to bind the same AT-rich regions of chromosomes and function to coordinate the control of a common set of genes.Here,we explored the molecular mechanism by which MvaU controls PCA biosynthesis in P.aeruginosa PA1201.We present evidence suggesting that MvaU is self-regulated.Deletion of mvaU significantly increased PCA production,and PCA production sharply decreased when mvaU was over-expressed.MvaU transcriptionally repressed phz2 cluster expression and consequently reduced PCA biosynthesis.β-galactosidase assays confirmed that base pairing near the35 box is required when MvaU regulates PCA production in PA1201.Electrophoretic mobility shift assays(EMSA)and additional point mutation analysis demonstrated that MvaU directly bound to an AT-rich motif within the promoter of the phz2 cluster.Chromatin immunoprecipitation(ChIP)analysis also indicated that MvaU directly bound to the P5 region of the phz2 cluster promoter.MvaU repression of PCA biosynthesis was independent of QscR and OxyR in PA1201 and neither PCA or H2O2 were the environmental signals that induced mvaU expression.These findings detail a new MvaU-dependent regulatory pathway of PCA biosynthesis in PA1201 and provide a foundation to increase PCA fermentation titer by genetic engineering.
基金funded by the Walmart Foundation(0402-70013-21-0000)supported by the Walmart Food Safety Collaboration Center
文摘Some veterinary drug residues in food products and environment have been widely regarded as severe threats to human health.Rapid and simultaneous detection methods are crucial to monitor and control veterinary drug usage.Here,we propose a fluorescence biosensor utilizing immunomagnetic beads(IMBs)and quantum dots(QDs)for the rapid and simultaneous detection of 1-adamantylamine(ADA),enrofloxacin(ENR)and tilmicosin(TIL)in raw chicken meat.A pretreatment method using sodium phosphotungstate–magnesium as extraction reagent was developed to simultaneously extract ADA,ENR and TIL from chicken meat with minor interference in background or response.By adding the IMBs modified with three types of antibodies and the QD-antigens modified with three types of BSA-antigens to sample,IMBs competitively conjugated to target antigens in a sample or QD-antigens.After magnetic separation,the residual QD-antigens were adopted to collect signals using fluorescence spectroscopy.Using QDs with well separated emission peaks,the detection of one type of targets was minorly interfered by the others.Under the optimum conditions,the biosensor exhibited the limit of detection of 0.96,3.32,and 3.17 ng/mL for ADA,ENR and TIL in chicken samples,respectively,as well as good specificity.Due to the way of direct collection of signals in extracts,the tedious and complicated multiple magnetic separation and signal amplification procedures in conventional methods were avoided,thus the procedures were significantly simplified,and the reduction of the operation time of 30 min for sample pretreatment and 40 min for detection part was achieved.The biosensor might be promising in the rapid,in-field and sensitive screening of multiple veterinary drugs to ensure agriculture and food safety.
