The pltR gene,coding a putative LysR-type regulator,was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp.M18 using bioinformatics technology.The null mutation of pltR resulted in mutant M18TRG(pltR:...The pltR gene,coding a putative LysR-type regulator,was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp.M18 using bioinformatics technology.The null mutation of pltR resulted in mutant M18TRG(pltR::Gm)by recombination and its Plt(Pyoluteorin)production declined to 30%while PCA(Phenazine-1-carboxylic acid)production remained unchanged as compared with the wild-type M18 grown in King’s Medium B.After complementation,Plt production of mutant M18TRG was restored to the level in wild-type M18.Overexpression of pltR in M18 led to 13-fold enhancement of Plt produc-tion over the wild-type M18 strain.However,PCA production was unchanged under this condition.These data suggested that PltR was a positive regulator on Plt production.Plt itself,however,could not regulate expression of pltR.Expression of the plt-lacZ transcriptional fusion in mutant M18TRG de-clined obviously as compared with the wild-type M18,which further proved that PltR could regulate expression of Plt biosynthetic genes at the transcriptional level.In addition,the investigation on the pltR expression in gacA mutant M18G and rsmA mutant M18R disclosed that PltR was involved in the positive regulation of gacA on Plt production while being excluded from the negative control caused by rsmA.展开更多
Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our in...Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16,located upstream of spinosad biosynthetic genes,in spinosad biosynthesis.Through reverse transcription PCR(RT-PCR)and 5′-rapid amplification of cDNA ends(5′-Race),we unveiled that the spinosad biosynthetic gene cluster(BGC)contains six transcription units and seven promoters.Electrophoretic mobility shift assays(EMSAs)demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC,indicating its involvement in regulating spinosad biosynthesis.Notably,deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L,accompanied by decreased transcription levels of spinosad biosynthetic genes,confirming its positive regulatory function.Additionally,isothermal titration calorimetry(ITC)and EMSA confirmed that spinosyn A,the main product of the spinosad BGC,served as an effector of ORF-L16.Specifically,it decreased the binding affinity between ORF-L16 and spinosad BGC promoters,thus exerting negative feedback regulation on spinosad biosynthesis.This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S.spinosa.展开更多
Lys R-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae,leading to severe infection.Earlier,we found a novel Lys R family gene,named kp053...Lys R-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae,leading to severe infection.Earlier,we found a novel Lys R family gene,named kp05372,in a strain of K.pneumoniae(designated GPKP)isolated from forest musk deer.To study the function of this gene in relation to the biological characteristics of GPKP,we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372;moreover,we also constructed the GPKP-Δkp05372+complemented strain.The role of this gene was determined by comparing the following characteristics of three strains:growth curves,biofilm formation,drug resistance,stress resistance,median lethal dose(LD50),organ colonization ability,and the histopathology of GPKP.Real-time polymerase chain reaction(RT-PCR)was used to test the expression level of seven genes upstream of kp05372.There was no significant difference in the growth rates when comparing the three bacterial strains,and no significant difference was recorded at different osmotic pressures,temperatures,salt contents,or hydrogen peroxide concentrations.The GPKP-Δkp05372 mutant formed a weak biofilm,and the other two strains formed medium biofilm.The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin,cotrimoxazole,and polymyxin B was changed.The acid tolerance of the deletion strain was stronger than that of the other two strains.The LD50 values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant,respectively.The colonization ability of the GPKP-Δkp05372 mutant in the heart,liver,spleen,kidney,and intestine was the weakest.The three strains caused different histopathological changes in the liver and lungs.In the GPKP-Δkp05372 mutant,the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold,respectively,while the level of kp05378 was decreased by 42%.Overall,the deletion of kp05372 gene leads to changes in the following:drug resistance and acid tolerance;decreases in virulence,biofilm formation,and colonization ability of GPKP;and regulation of the upstream region of adjacent genes.展开更多
GcvA is a member of the LysR family of transcriptional regulators that mediates the expression of the glycine cleavage (GCV) operon (gcvTHP) in response to glycine in Escherichia coli. In our previous work, 90 putativ...GcvA is a member of the LysR family of transcriptional regulators that mediates the expression of the glycine cleavage (GCV) operon (gcvTHP) in response to glycine in Escherichia coli. In our previous work, 90 putative regulator genes of the LysR family in Sinorhizobium meliloti were mutagenized to determine their phenotype. In the present study, we found that the S. meliloti genome had two gcvA genes, gcvA1 and gcvA2. Both gcvA1 and gcvA2 were required for full activation of the gcvTHP operon in the presence of exogenous glycine. The gcvA1-mediated activation of gcvTHP operon was gly- cine-inducible, while gcvA2-mediated activation was not. We speculate that the regulatory mechanism for gcvTHP expression in S. meliloti differed from E. coli. Evolutionary analysis showed that GcvA were distributed in many genera of Proteobacteria and the distances between GcvA1 and GcvA2 in S. meliloti and GcvA in E. coli were large, which may explain the different regulatory mechanisms for gcvTHP expression. These findings could provide new clues to the role of the LysR gene family.展开更多
基金Supported by the Science Program of the China National 10th"Five-year Plan"(Grant No.2004BA308A21-6)the National Natural Science Foundation of China(Grant No.30370041)and NCET in China
文摘The pltR gene,coding a putative LysR-type regulator,was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp.M18 using bioinformatics technology.The null mutation of pltR resulted in mutant M18TRG(pltR::Gm)by recombination and its Plt(Pyoluteorin)production declined to 30%while PCA(Phenazine-1-carboxylic acid)production remained unchanged as compared with the wild-type M18 grown in King’s Medium B.After complementation,Plt production of mutant M18TRG was restored to the level in wild-type M18.Overexpression of pltR in M18 led to 13-fold enhancement of Plt produc-tion over the wild-type M18 strain.However,PCA production was unchanged under this condition.These data suggested that PltR was a positive regulator on Plt production.Plt itself,however,could not regulate expression of pltR.Expression of the plt-lacZ transcriptional fusion in mutant M18TRG de-clined obviously as compared with the wild-type M18,which further proved that PltR could regulate expression of Plt biosynthetic genes at the transcriptional level.In addition,the investigation on the pltR expression in gacA mutant M18G and rsmA mutant M18R disclosed that PltR was involved in the positive regulation of gacA on Plt production while being excluded from the negative control caused by rsmA.
基金supported by the National Key R&D Program of China(grant number 2018YFA0900400)the National Natural Science Foundation of China(grant number 32100053)the Young Elite Scientists Sponsorship Program by China Association for Science and Technology(grant number YESS20210068).
文摘Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16,located upstream of spinosad biosynthetic genes,in spinosad biosynthesis.Through reverse transcription PCR(RT-PCR)and 5′-rapid amplification of cDNA ends(5′-Race),we unveiled that the spinosad biosynthetic gene cluster(BGC)contains six transcription units and seven promoters.Electrophoretic mobility shift assays(EMSAs)demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC,indicating its involvement in regulating spinosad biosynthesis.Notably,deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L,accompanied by decreased transcription levels of spinosad biosynthetic genes,confirming its positive regulatory function.Additionally,isothermal titration calorimetry(ITC)and EMSA confirmed that spinosyn A,the main product of the spinosad BGC,served as an effector of ORF-L16.Specifically,it decreased the binding affinity between ORF-L16 and spinosad BGC promoters,thus exerting negative feedback regulation on spinosad biosynthesis.This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S.spinosa.
基金Project supported by the Sichuan Province Academic and Technical Leadership Development Funding Project and the Science&Technology Achievements Transfer Project of Sichuan(No.2017YSZH0008),China.
文摘Lys R-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae,leading to severe infection.Earlier,we found a novel Lys R family gene,named kp05372,in a strain of K.pneumoniae(designated GPKP)isolated from forest musk deer.To study the function of this gene in relation to the biological characteristics of GPKP,we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372;moreover,we also constructed the GPKP-Δkp05372+complemented strain.The role of this gene was determined by comparing the following characteristics of three strains:growth curves,biofilm formation,drug resistance,stress resistance,median lethal dose(LD50),organ colonization ability,and the histopathology of GPKP.Real-time polymerase chain reaction(RT-PCR)was used to test the expression level of seven genes upstream of kp05372.There was no significant difference in the growth rates when comparing the three bacterial strains,and no significant difference was recorded at different osmotic pressures,temperatures,salt contents,or hydrogen peroxide concentrations.The GPKP-Δkp05372 mutant formed a weak biofilm,and the other two strains formed medium biofilm.The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin,cotrimoxazole,and polymyxin B was changed.The acid tolerance of the deletion strain was stronger than that of the other two strains.The LD50 values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant,respectively.The colonization ability of the GPKP-Δkp05372 mutant in the heart,liver,spleen,kidney,and intestine was the weakest.The three strains caused different histopathological changes in the liver and lungs.In the GPKP-Δkp05372 mutant,the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold,respectively,while the level of kp05378 was decreased by 42%.Overall,the deletion of kp05372 gene leads to changes in the following:drug resistance and acid tolerance;decreases in virulence,biofilm formation,and colonization ability of GPKP;and regulation of the upstream region of adjacent genes.
基金Supported by National Basic Research and Development Program of China (Grant No. 2001CB108901)USA NIH (5S06GM8225) PSC-CUNY (617320030 and 632140032)
文摘GcvA is a member of the LysR family of transcriptional regulators that mediates the expression of the glycine cleavage (GCV) operon (gcvTHP) in response to glycine in Escherichia coli. In our previous work, 90 putative regulator genes of the LysR family in Sinorhizobium meliloti were mutagenized to determine their phenotype. In the present study, we found that the S. meliloti genome had two gcvA genes, gcvA1 and gcvA2. Both gcvA1 and gcvA2 were required for full activation of the gcvTHP operon in the presence of exogenous glycine. The gcvA1-mediated activation of gcvTHP operon was gly- cine-inducible, while gcvA2-mediated activation was not. We speculate that the regulatory mechanism for gcvTHP expression in S. meliloti differed from E. coli. Evolutionary analysis showed that GcvA were distributed in many genera of Proteobacteria and the distances between GcvA1 and GcvA2 in S. meliloti and GcvA in E. coli were large, which may explain the different regulatory mechanisms for gcvTHP expression. These findings could provide new clues to the role of the LysR gene family.
