蛋白质硫辛酸修饰相关酶大肠杆菌研究模型的构建
|
摘要
为研究不同微生物硫辛酸修饰相关酶的功能,构建了大肠杆菌菌体内研究模型。利用Red同源重组系统对大肠杆菌的lplA、lipB基因依次进行敲除,获得大肠杆菌lplA及lipB双基因缺失株△lplA△lipB～DH5α。将lplA和lipB基因分别克隆到载体pBAD322G,构建基因回补株ClplA-△lplA△lipB～DH5α和ClipB-△lplA△lipB～DH5α。通过Western blot、质谱分析和补偿试验等方法检测缺失株和回补株对底物的硫辛酸修饰功能。结果显示:上述突变株构建成功并能稳定遗传,回补株构建成功并能发挥生物学功能。补偿试验结果显示,在甘油为碳源的M9基础培养基中,回补株ClipB-△lplA△lipB～DH5α能够生长, ClplA-△lplA△lipB～DH5α补偿硫辛酸后能够生长。以上结果表明,大肠杆菌DH5α硫辛酸修饰功能缺陷菌株构建成功,这有利于以大肠杆菌为生物模型进行其他微生物硫辛酸修饰相关酶的功能研究。
To investigate the function of lipoic acid metabolic enzymes in vivo, an Escherichia coli(E. coli) model was established.△lplA△lipB～DH5α mutant strain was obtained with deletion of lplA and lipB genes from DH5α strain using the Red homologous recombination system. The lplA and lipB genes were cloned into the expression vector of pBAD322 G and transformed to the △lplA△lipB～DH5α mutant strain to construct the complementary strains of ClplA-△lplA△lipB～DH5α and ClipB-△lplA△lipB～DH5α. The lipoate modification on the substrates in the mutant and supplemented strains were analyzed with Western blot, mass spectrometric analysis and complementation experiment. The mutant strain △lplA△lipB～DH5α was very stable in the culture and displayed the phenotype of lplA and lipB deletion. Supplemented strain ClipB-△lplA△lipB～DH5α can grow in the M9 medium with glycerol as carbon source, ClplA-△lplA△lipB～DH5α can grow when LA is added to this medium. These results indicated that the E. coli model without lipoate modification function was successfully established. It will facilitate the functional study of enzymes associated with lipoic acid modification in other microorganism.
To investigate the function of lipoic acid metabolic enzymes in vivo, an Escherichia coli(E. coli) model was established.△lplA△lipB～DH5α mutant strain was obtained with deletion of lplA and lipB genes from DH5α strain using the Red homologous recombination system. The lplA and lipB genes were cloned into the expression vector of pBAD322 G and transformed to the △lplA△lipB～DH5α mutant strain to construct the complementary strains of ClplA-△lplA△lipB～DH5α and ClipB-△lplA△lipB～DH5α. The lipoate modification on the substrates in the mutant and supplemented strains were analyzed with Western blot, mass spectrometric analysis and complementation experiment. The mutant strain △lplA△lipB～DH5α was very stable in the culture and displayed the phenotype of lplA and lipB deletion. Supplemented strain ClipB-△lplA△lipB～DH5α can grow in the M9 medium with glycerol as carbon source, ClplA-△lplA△lipB～DH5α can grow when LA is added to this medium. These results indicated that the E. coli model without lipoate modification function was successfully established. It will facilitate the functional study of enzymes associated with lipoic acid modification in other microorganism.
引文
[1] REED L J.A trail of research from lipoic acid to α-keto acid dehydrogenase complexes[J].J Biol Chem,2001,276(42):38329-38336.
[2] O′RIORDAN M,MOORS M A,PORTNOY D A.Listeria intracellular growth and virulence require host-derived lipoic acid[J].Science,2003,302(5644):462-464.
[3] 贺华良,宾淑英,廖泓之,等.黄曲条跳甲线粒体源硫辛酸蛋白连接酶的序列分析及表达[J].华南农业大学学报,2012,33(3):351-355.HE H L,BIN S Y,LIAO H Z,et al.Sequencing and expression profile of the gene encodinglipoate protein ligase in Phyllotreta striolata[J].Journal of South China Agricultural University,2012,33(3):351-355.(in Chinese)
[4] MORRIS T W,REED K E,CRONAN J E Jr.Identification of the gene encoding lipoate-protein ligase A of Escherichia coli.Molecular cloning and characterization of the lplA gene and gene product[J].J Biol Chem,1994,269(23):16091-16100.
[5] MORRIS T W,REED K E,CRONAN J E Jr.Lipoic acid metabolism in Escherichia coli:the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein[J].J Bacteriol,1995,177(1):1-10.
[6] CHRISTENSEN Q H,CRONAN J E.Lipoic acid synthesis:a new family of octanoyltransferases generally annotated as lipoate protein ligases[J].Biochemistry,2010,49(46):10024-10036.
[7] 于艳超,王莉莉,潘巧,等.肠炎沙门菌luxS缺失株的构建及其生物学特性研究[J].畜牧兽医学报,2018,49(6):1291-1298.YU Y C,WANG L L,PAN Q,et al.Construction and biological characteristics of Salmonella enterica serovar enteritidis luxS mutant[J].Acta Veterinaria et Zootechnica Sinica,2018,49(6):1291-1298.(in Chinese)
[8] 苏洋洋,黄骏,吴白,等.鼠伤寒沙门菌rpoN基因缺失株构建及生物学特性研究[J].畜牧兽医学报,2016,47(4):771-778.SU Y Y,HUANG J,WU B,et al.Construction and biological characterization of rpoN gene deletion mutants of Salmonella typhimurium[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):771-778.(in Chinese)
[9] CHAKRAVARTTY V,CRONAN J E.A series of medium and high copy number arabinose-inducible Escherichia coli expression vectors compatible with pBR322 and pACYC184[J].Plasmid,2015,81:21-26.
[10] BERG M,SEEMüLLER E.Chromosomal organization and nucleotide sequence of the genes coding for the elongation factors G and Tu of the apple proliferation phytoplasma[J].Gene,1999,226(1):103-109.
[11] 司微,于申业,陈利苹,等.肠炎沙门氏菌tu-fA基因缺失株的构建及其生物学特性研究[J].中国预防兽医学报,2015,37(5):361-365.SI W,YU S Y,CHEN L P,et al.Construction and biological identification of a tu-fA gene deletion mutant from Salmonella enterica[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(5):361-365.(in Chinese)
[12] CASTUMA C E,HUANG R P,KORNBERG A,et al.Inorganic polyphosphates in the acquisition of competence in Escherichia coli[J].J Biol Chem,1995,270(22):12980-12983.
[13] FUJIWARA K,MAITA N,HOSAKA H,et al.Global Conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A[J].J Biol Chem,2010,285(13):9971-9980.
[14] CAO X Y,CRONAN J E.The Streptomyces coelicolor lipoate-protein ligase is a circularly permuted version of the Escherichia coli enzyme composed of discrete interacting domains[J].J Biol Chem,2015,290(11):7280-7290.
[15] CRONAN J E.A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control[J].Plasmid,2006,55(2):152-157.
[16] CRONAN J E,ZHAO X,JIANG Y F.Function,attachment and synthesis of lipoic acid in Escherichia coli[J].Adv Micro Physiol,2005,50:103-146.
[17] PERHAM R N.Swinging arms and swinging domains in multifunctional enzymes:catalytic machines for multistep reactions[J].Ann Rev Biochem,2000,69(69):961-1004.
[18] 杨晓亮,尹楠林,庞丹,等.假想的脂蛋白连接酶对肺炎链球菌毒力的影响[J].微生物学报,2010,50(6):774-779.YANG X L,YIN N L,PANG D,et al.Contributions of putative lipoate-protein ligase to the virulence of Streptococcus pneumoniae[J].Acta Microbiologica Sinica,2010,50(6):774-779.(in Chinese)
[19] 黄涛,黄开勋.硫辛酸修饰胰岛素与胰岛素受体的结合能力[J].中国生化药物杂志,2006,27(2):65-68.HUANG T,HUANG K X.The binding potency oflipoic acid modified insulin with insulin receptor[J].Chinese Journal of Biochemical Pharmaceutics,2006,27(2):65-68.(in Chinese)
[20] BRYK R,LIMA C D,ERDJUMENT-BROMAGE H,et al.Metabolic enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein[J].Science,2002,295(5557):1073-1077.
[21] OKAMURA-IKEDA K,HOSAKA H,MAITA N,et al.Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system:implications for recognition of lipoyl protein substrate,disease-related mutations,and reaction mechanism[J].J Biol Chem,2010,285(24):18684-18692.
[2] O′RIORDAN M,MOORS M A,PORTNOY D A.Listeria intracellular growth and virulence require host-derived lipoic acid[J].Science,2003,302(5644):462-464.
[3] 贺华良,宾淑英,廖泓之,等.黄曲条跳甲线粒体源硫辛酸蛋白连接酶的序列分析及表达[J].华南农业大学学报,2012,33(3):351-355.HE H L,BIN S Y,LIAO H Z,et al.Sequencing and expression profile of the gene encodinglipoate protein ligase in Phyllotreta striolata[J].Journal of South China Agricultural University,2012,33(3):351-355.(in Chinese)
[4] MORRIS T W,REED K E,CRONAN J E Jr.Identification of the gene encoding lipoate-protein ligase A of Escherichia coli.Molecular cloning and characterization of the lplA gene and gene product[J].J Biol Chem,1994,269(23):16091-16100.
[5] MORRIS T W,REED K E,CRONAN J E Jr.Lipoic acid metabolism in Escherichia coli:the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein[J].J Bacteriol,1995,177(1):1-10.
[6] CHRISTENSEN Q H,CRONAN J E.Lipoic acid synthesis:a new family of octanoyltransferases generally annotated as lipoate protein ligases[J].Biochemistry,2010,49(46):10024-10036.
[7] 于艳超,王莉莉,潘巧,等.肠炎沙门菌luxS缺失株的构建及其生物学特性研究[J].畜牧兽医学报,2018,49(6):1291-1298.YU Y C,WANG L L,PAN Q,et al.Construction and biological characteristics of Salmonella enterica serovar enteritidis luxS mutant[J].Acta Veterinaria et Zootechnica Sinica,2018,49(6):1291-1298.(in Chinese)
[8] 苏洋洋,黄骏,吴白,等.鼠伤寒沙门菌rpoN基因缺失株构建及生物学特性研究[J].畜牧兽医学报,2016,47(4):771-778.SU Y Y,HUANG J,WU B,et al.Construction and biological characterization of rpoN gene deletion mutants of Salmonella typhimurium[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):771-778.(in Chinese)
[9] CHAKRAVARTTY V,CRONAN J E.A series of medium and high copy number arabinose-inducible Escherichia coli expression vectors compatible with pBR322 and pACYC184[J].Plasmid,2015,81:21-26.
[10] BERG M,SEEMüLLER E.Chromosomal organization and nucleotide sequence of the genes coding for the elongation factors G and Tu of the apple proliferation phytoplasma[J].Gene,1999,226(1):103-109.
[11] 司微,于申业,陈利苹,等.肠炎沙门氏菌tu-fA基因缺失株的构建及其生物学特性研究[J].中国预防兽医学报,2015,37(5):361-365.SI W,YU S Y,CHEN L P,et al.Construction and biological identification of a tu-fA gene deletion mutant from Salmonella enterica[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(5):361-365.(in Chinese)
[12] CASTUMA C E,HUANG R P,KORNBERG A,et al.Inorganic polyphosphates in the acquisition of competence in Escherichia coli[J].J Biol Chem,1995,270(22):12980-12983.
[13] FUJIWARA K,MAITA N,HOSAKA H,et al.Global Conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A[J].J Biol Chem,2010,285(13):9971-9980.
[14] CAO X Y,CRONAN J E.The Streptomyces coelicolor lipoate-protein ligase is a circularly permuted version of the Escherichia coli enzyme composed of discrete interacting domains[J].J Biol Chem,2015,290(11):7280-7290.
[15] CRONAN J E.A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control[J].Plasmid,2006,55(2):152-157.
[16] CRONAN J E,ZHAO X,JIANG Y F.Function,attachment and synthesis of lipoic acid in Escherichia coli[J].Adv Micro Physiol,2005,50:103-146.
[17] PERHAM R N.Swinging arms and swinging domains in multifunctional enzymes:catalytic machines for multistep reactions[J].Ann Rev Biochem,2000,69(69):961-1004.
[18] 杨晓亮,尹楠林,庞丹,等.假想的脂蛋白连接酶对肺炎链球菌毒力的影响[J].微生物学报,2010,50(6):774-779.YANG X L,YIN N L,PANG D,et al.Contributions of putative lipoate-protein ligase to the virulence of Streptococcus pneumoniae[J].Acta Microbiologica Sinica,2010,50(6):774-779.(in Chinese)
[19] 黄涛,黄开勋.硫辛酸修饰胰岛素与胰岛素受体的结合能力[J].中国生化药物杂志,2006,27(2):65-68.HUANG T,HUANG K X.The binding potency oflipoic acid modified insulin with insulin receptor[J].Chinese Journal of Biochemical Pharmaceutics,2006,27(2):65-68.(in Chinese)
[20] BRYK R,LIMA C D,ERDJUMENT-BROMAGE H,et al.Metabolic enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein[J].Science,2002,295(5557):1073-1077.
[21] OKAMURA-IKEDA K,HOSAKA H,MAITA N,et al.Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system:implications for recognition of lipoyl protein substrate,disease-related mutations,and reaction mechanism[J].J Biol Chem,2010,285(24):18684-18692.