Biochemical data and genomic analysis indicate the involvement of a putative ABC transporter for glucose transport in Pseudomonas putida CSV86. The periplasmic solute binding proteins are known to confer substrate spe...Biochemical data and genomic analysis indicate the involvement of a putative ABC transporter for glucose transport in Pseudomonas putida CSV86. The periplasmic solute binding proteins are known to confer substrate specificity to the ABC transporters by binding specifically to the substrate and transferring them to their cognate inner membrane transport assembly. Periplasmic glucose binding protein from Pseudomonas putida CSV86 (ppGBP) was found to be glucose specific. The gene encoding ppGBP was cloned. Recombinant ppGBP was overexpressed and purified to homogeneity. The purified recombinant protein showed glucose binding activity of 752 pmol/mg of protein and was crystallized as a complex with glucose. The crystal diffracted to 1.7 Å resolution using home X-ray source. Preliminary analysis of diffraction data showed that the crystals belonged to space group P21212 with unit-cell parameters a = 102.56, b = 119.2, c = 66.65 Å and α = β = γ = 90°. Matthews coefficient calculation showed the presence of two molecules in the asymmetric unit with solvent content of 45.7%.展开更多
Reverse-transcription qualitative PCR(RT-qPCR)was used to analyze the changes in transcription levels of the sulfur metabolism-related periplasmic protein genes of Acidithiobacillus ferrooxidans ATCC 23270 grown on su...Reverse-transcription qualitative PCR(RT-qPCR)was used to analyze the changes in transcription levels of the sulfur metabolism-related periplasmic protein genes of Acidithiobacillus ferrooxidans ATCC 23270 grown on sulfur or ferrous.Seven periplasmic proteins with apparently higher abundance grown on elemental sulfur than on ferrous sulfate were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS).Expression analysis of the corresponding genes by RT-qPCR shows that the constitutive expression of all those genes are more up-regulated grown on sulfur than those grown on ferrous(>10 folder).Study on the corresponding genes of the identified periplasmic proteins by RT-qPCR confirmed the results of two-dimensioned gel electrophoresis,indicating they may be related with sulfur metabolism in A.ferrooxidans.展开更多
为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能,对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列,利用PCR方法扩增到该基因的编码区序列,利用SLIC(Sequence and lig...为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能,对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列,利用PCR方法扩增到该基因的编码区序列,利用SLIC(Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中,成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达,通过改变培养温度和IPTG浓度等条件,得到了能够大量表达目标蛋白的重组子。结果表明:大肠杆菌BL21(DE3)是gsiB基因表达的最佳宿主菌;18℃低温诱导培养有利于gsiB基因的大量表达;0.1mmol/LIPTG足够诱导gsiB基因表达,增加IPTG浓度(0.1mmol/L～1.0mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达,其分子量大小与预期相符。展开更多
The L-forms were induced from Staphylococcus aureus,Escherichia coli and Bacdtos cereus by β-lactam anhbiohcs and then observahons on the proPenies of oxygen requlrement sugar fermentahon and sensihve tO cyanide of t...The L-forms were induced from Staphylococcus aureus,Escherichia coli and Bacdtos cereus by β-lactam anhbiohcs and then observahons on the proPenies of oxygen requlrement sugar fermentahon and sensihve tO cyanide of the Lforms were done. The resultS were shown that the Lforms derived from the obligate aerobe or the faCultative anaerobe did not ferment sugars and were highly oxygendePendent and more sensihve tD cyedde than their Parent bacteria The metabolic achvihes which were same as the Parent bacteria of the Lforms would retUrn after the Lforms reveded to the typical bacterial forms. It was possible that the changes of biological oxidation mechasm were due to the deficiency of the cell wall which led to loss of the periplasndc space or the membr-anetwall interiayer so that the enzymes for fermentation existed in the space could not be hold.展开更多
Proteins secreted by Helicobacter pylori (H. pylori), an important human pathogen responsible for severe gastric diseases, are reviewed from the point of view of their biochemical characterization, both functional and...Proteins secreted by Helicobacter pylori (H. pylori), an important human pathogen responsible for severe gastric diseases, are reviewed from the point of view of their biochemical characterization, both functional and structural. Despite the vast amount of experimental data available on the proteins secreted by this bacterium, the precise size of the secretome remains unknown. In this review, we consider as secreted both proteins that contain a secretion signal for the periplasm and proteins that have been detected in the external medium in in vitro experiments. In this way, H. pylori’s secretome appears to be composed of slightly more than 160 proteins, but this number must be considered very cautiously, not only because the definition of secretome itself is ambiguous but also because the included proteins were observed as secreted in in vitro experiments that were not representative of the environmental situation in vivo. The proteins that appear to be secreted can be grouped into different classes: enzymes (48 proteins), outer membrane proteins (43), components of flagella (11), members of the cytotoxic-associated genes pathogenicity island or other toxins (8 and 5, respectively), binding and transport proteins (9), and others (11). A final group, which includes 28 members, is represented by hypothetical uncharacterized proteins. Despite the large amount of data accumulated on the H. pylori secretome, a considerable amount of work remains to reach a full comprehension of the system at the molecular level.展开更多
文摘Biochemical data and genomic analysis indicate the involvement of a putative ABC transporter for glucose transport in Pseudomonas putida CSV86. The periplasmic solute binding proteins are known to confer substrate specificity to the ABC transporters by binding specifically to the substrate and transferring them to their cognate inner membrane transport assembly. Periplasmic glucose binding protein from Pseudomonas putida CSV86 (ppGBP) was found to be glucose specific. The gene encoding ppGBP was cloned. Recombinant ppGBP was overexpressed and purified to homogeneity. The purified recombinant protein showed glucose binding activity of 752 pmol/mg of protein and was crystallized as a complex with glucose. The crystal diffracted to 1.7 Å resolution using home X-ray source. Preliminary analysis of diffraction data showed that the crystals belonged to space group P21212 with unit-cell parameters a = 102.56, b = 119.2, c = 66.65 Å and α = β = γ = 90°. Matthews coefficient calculation showed the presence of two molecules in the asymmetric unit with solvent content of 45.7%.
基金Projects(50674101,50974140)supported by the National Natural Science Foundation of ChinaProject(2010CB630902)supported by the National Basic Research Program of China
文摘Reverse-transcription qualitative PCR(RT-qPCR)was used to analyze the changes in transcription levels of the sulfur metabolism-related periplasmic protein genes of Acidithiobacillus ferrooxidans ATCC 23270 grown on sulfur or ferrous.Seven periplasmic proteins with apparently higher abundance grown on elemental sulfur than on ferrous sulfate were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS).Expression analysis of the corresponding genes by RT-qPCR shows that the constitutive expression of all those genes are more up-regulated grown on sulfur than those grown on ferrous(>10 folder).Study on the corresponding genes of the identified periplasmic proteins by RT-qPCR confirmed the results of two-dimensioned gel electrophoresis,indicating they may be related with sulfur metabolism in A.ferrooxidans.
文摘为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能,对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列,利用PCR方法扩增到该基因的编码区序列,利用SLIC(Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中,成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达,通过改变培养温度和IPTG浓度等条件,得到了能够大量表达目标蛋白的重组子。结果表明:大肠杆菌BL21(DE3)是gsiB基因表达的最佳宿主菌;18℃低温诱导培养有利于gsiB基因的大量表达;0.1mmol/LIPTG足够诱导gsiB基因表达,增加IPTG浓度(0.1mmol/L～1.0mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达,其分子量大小与预期相符。
文摘The L-forms were induced from Staphylococcus aureus,Escherichia coli and Bacdtos cereus by β-lactam anhbiohcs and then observahons on the proPenies of oxygen requlrement sugar fermentahon and sensihve tO cyanide of the Lforms were done. The resultS were shown that the Lforms derived from the obligate aerobe or the faCultative anaerobe did not ferment sugars and were highly oxygendePendent and more sensihve tD cyedde than their Parent bacteria The metabolic achvihes which were same as the Parent bacteria of the Lforms would retUrn after the Lforms reveded to the typical bacterial forms. It was possible that the changes of biological oxidation mechasm were due to the deficiency of the cell wall which led to loss of the periplasndc space or the membr-anetwall interiayer so that the enzymes for fermentation existed in the space could not be hold.
基金Supported by the University of Padua grant "Progetto di Ateneo 2011" and by PRIN 2010-2011 "Unraveling structural and functional determinants behind Helicobacter pylori pathogen-esis and persistence"
文摘Proteins secreted by Helicobacter pylori (H. pylori), an important human pathogen responsible for severe gastric diseases, are reviewed from the point of view of their biochemical characterization, both functional and structural. Despite the vast amount of experimental data available on the proteins secreted by this bacterium, the precise size of the secretome remains unknown. In this review, we consider as secreted both proteins that contain a secretion signal for the periplasm and proteins that have been detected in the external medium in in vitro experiments. In this way, H. pylori’s secretome appears to be composed of slightly more than 160 proteins, but this number must be considered very cautiously, not only because the definition of secretome itself is ambiguous but also because the included proteins were observed as secreted in in vitro experiments that were not representative of the environmental situation in vivo. The proteins that appear to be secreted can be grouped into different classes: enzymes (48 proteins), outer membrane proteins (43), components of flagella (11), members of the cytotoxic-associated genes pathogenicity island or other toxins (8 and 5, respectively), binding and transport proteins (9), and others (11). A final group, which includes 28 members, is represented by hypothetical uncharacterized proteins. Despite the large amount of data accumulated on the H. pylori secretome, a considerable amount of work remains to reach a full comprehension of the system at the molecular level.
