The oligotrophic freshwater bacterium Caulobacter crescentus encodes a cluster of genes (CC_1487 to CC_1495) shown here to be necessary for metabolism of D-galacturonate, the primary constituent of pectin, a major pla...The oligotrophic freshwater bacterium Caulobacter crescentus encodes a cluster of genes (CC_1487 to CC_1495) shown here to be necessary for metabolism of D-galacturonate, the primary constituent of pectin, a major plant polymer. Sequence analysis suggests that these genes encode a version of the bacterial hexuronate isomerase pathway. A conserved 14 bp sequence motif is associated with promoter regions of three operons within this cluster, and is conserved in homologous gene clusters in related alpha-Proteobacteria. Embedded in the hexuronate gene cluster is a gene (CC_1489) encoding a member of the LacI family of bacterial transcription factors. This gene product, designated here as HumR (hexuronate metabolism regulator), represses expression of the uxaA and uxaC operon promoters by binding to the conserved operator sequence. Repression is relieved in the presence of galacturonate or, to a lesser extent, by glucuronate. Other genes potentially involved in pectin degradation and hexuronate transport are also under the control of HumR. Adoption of a LacI-type repressor to control hexuronate metabolism parallels the regulation of xylose, glucose, and maltose utilization in C. crescentus, but is distinct from the use of GntR-type repressors to control pectin and hexuronate utilization in gamma-Proteobacteria such as Escherichia coli.展开更多
Objective: To establish transgenic mouse lineages containing copies of a stably integratedpSPORT1 plasmid by microinjection into fertilized eggs and to provide an efficient animal model for studyinggene mutations in v...Objective: To establish transgenic mouse lineages containing copies of a stably integratedpSPORT1 plasmid by microinjection into fertilized eggs and to provide an efficient animal model for studyinggene mutations in vivo. Methods and Results: 2594 fertilized eggs from KM white mice were injected withpSPORT1 DNA and transferred into the oviducts of 103 pseudopregnant females. from which 237 offspringswere obtained. 40 of these offsprings were identified positive for the forgeign gene by PCR analysis and 38were reproved positive by Southern blot analysis. Finally, eight of the stout mice whose genornes were integrated with intact pSPORT1 vectors, verified by Southern blotting analysis, were chosen as founders to establish the transgenic mouse lineages. The experimental results inchoated that the pregnant rate of recipientfemales and the Positive rate of offsprings were dramatically influenced by the structure of the transgene(linearized or circular ) and the mode of egg-transfer. The integration rate of linearized transgene was significantly higher than that of the circular transgene,and female with two-side oviduct transfer of fertilized eggs waseasy to have baby mice. Conclusion: (1 ) Transgenic mouse lineages containing copies of a stably integratedPSPORT 1 plasmid were established; (2 ) The linearized transgene and two-side oviduct transfer of fertilizedeggs is more efficient in preparing transgenic mice.展开更多
Intracellular pH critically affects various biological processes,and an appropriate cytoplasmic pH is essential for ensuring bacterial growth.Glucose is the preferred carbon source for most heterotrophs;however,excess...Intracellular pH critically affects various biological processes,and an appropriate cytoplasmic pH is essential for ensuring bacterial growth.Glucose is the preferred carbon source for most heterotrophs;however,excess glucose often causes the accumulation of acidic metabolites,lowering the intracellular pH and inhibiting bacterial growth.Bacillus thuringiensis can effectively cope with glucose-induced stress;unfortunately,little is known about the regulators involved in this process.Here,we document that the target of the dual-function sRNA YhfH,the lipR gene,encodes a LacI-family transcription factor LipR as an intracellular pH regulator when B.thuringiensis BMB171 is suddenly exposed to glucose.Under glucose conditions,lipR deletion leads to early growth arrest by causing a rapid decrease in intracellular pH(~5.4).Then,the direct targets and a binding motif(GAWAWCRWTWTCAT)of LipR were identified based on the electrophoretic mobility shift assay,the DNase-I footprinting assay,and RNA sequencing,and the gapN gene encoding a key enzyme in glycolysis was directly inhibited by LipR.Furthermore,Ni^(2+)is considered a possible effector for LipR.In addition to YhfH,the lipR expression was coregulated by itself,CcpA,and AbrB.Our study reveals that LipR plays a balancing role between glucose metabolism and intracellular pH in B.thuringiensis subjected to glucose stress.展开更多
文摘The oligotrophic freshwater bacterium Caulobacter crescentus encodes a cluster of genes (CC_1487 to CC_1495) shown here to be necessary for metabolism of D-galacturonate, the primary constituent of pectin, a major plant polymer. Sequence analysis suggests that these genes encode a version of the bacterial hexuronate isomerase pathway. A conserved 14 bp sequence motif is associated with promoter regions of three operons within this cluster, and is conserved in homologous gene clusters in related alpha-Proteobacteria. Embedded in the hexuronate gene cluster is a gene (CC_1489) encoding a member of the LacI family of bacterial transcription factors. This gene product, designated here as HumR (hexuronate metabolism regulator), represses expression of the uxaA and uxaC operon promoters by binding to the conserved operator sequence. Repression is relieved in the presence of galacturonate or, to a lesser extent, by glucuronate. Other genes potentially involved in pectin degradation and hexuronate transport are also under the control of HumR. Adoption of a LacI-type repressor to control hexuronate metabolism parallels the regulation of xylose, glucose, and maltose utilization in C. crescentus, but is distinct from the use of GntR-type repressors to control pectin and hexuronate utilization in gamma-Proteobacteria such as Escherichia coli.
文摘Objective: To establish transgenic mouse lineages containing copies of a stably integratedpSPORT1 plasmid by microinjection into fertilized eggs and to provide an efficient animal model for studyinggene mutations in vivo. Methods and Results: 2594 fertilized eggs from KM white mice were injected withpSPORT1 DNA and transferred into the oviducts of 103 pseudopregnant females. from which 237 offspringswere obtained. 40 of these offsprings were identified positive for the forgeign gene by PCR analysis and 38were reproved positive by Southern blot analysis. Finally, eight of the stout mice whose genornes were integrated with intact pSPORT1 vectors, verified by Southern blotting analysis, were chosen as founders to establish the transgenic mouse lineages. The experimental results inchoated that the pregnant rate of recipientfemales and the Positive rate of offsprings were dramatically influenced by the structure of the transgene(linearized or circular ) and the mode of egg-transfer. The integration rate of linearized transgene was significantly higher than that of the circular transgene,and female with two-side oviduct transfer of fertilized eggs waseasy to have baby mice. Conclusion: (1 ) Transgenic mouse lineages containing copies of a stably integratedPSPORT 1 plasmid were established; (2 ) The linearized transgene and two-side oviduct transfer of fertilizedeggs is more efficient in preparing transgenic mice.
基金supported by the National Key Research and Development Program of China(2018YFA0900100)the National Natural Science Foundation of China(31670081).
文摘Intracellular pH critically affects various biological processes,and an appropriate cytoplasmic pH is essential for ensuring bacterial growth.Glucose is the preferred carbon source for most heterotrophs;however,excess glucose often causes the accumulation of acidic metabolites,lowering the intracellular pH and inhibiting bacterial growth.Bacillus thuringiensis can effectively cope with glucose-induced stress;unfortunately,little is known about the regulators involved in this process.Here,we document that the target of the dual-function sRNA YhfH,the lipR gene,encodes a LacI-family transcription factor LipR as an intracellular pH regulator when B.thuringiensis BMB171 is suddenly exposed to glucose.Under glucose conditions,lipR deletion leads to early growth arrest by causing a rapid decrease in intracellular pH(~5.4).Then,the direct targets and a binding motif(GAWAWCRWTWTCAT)of LipR were identified based on the electrophoretic mobility shift assay,the DNase-I footprinting assay,and RNA sequencing,and the gapN gene encoding a key enzyme in glycolysis was directly inhibited by LipR.Furthermore,Ni^(2+)is considered a possible effector for LipR.In addition to YhfH,the lipR expression was coregulated by itself,CcpA,and AbrB.Our study reveals that LipR plays a balancing role between glucose metabolism and intracellular pH in B.thuringiensis subjected to glucose stress.
