谷氨酸棒状杆菌ATCC 14067阻遏蛋白ArgR与L-精氨酸合成途径相关基因的相互作用
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摘要
在细菌中阻遏蛋白ArgR通过与精氨酸合成途径相关基因中的保守序列结合,阻遏相关基因的转录。在谷氨酸棒状杆菌ATCC 14067中,精氨酸生物合成涉及的基因位于基因簇argCJBDFRGH和carAB中。构建重组菌株E.coli BL21(DE3)/pET22b-argR,诱导表达ATCC 14067来源的ArgR,成功得到目的蛋白。利用分子筛色谱柱对ArgR蛋白进行分析,表明ArgR蛋白相对分子质量在108 ku左右,在活性状态下ArgR蛋白为六聚体。合成生物素标记的谷氨酸棒状杆菌ATCC 14067来源的argC、argJ、argB、argF、argG、argH、carA基因片段,采用凝胶阻滞实验(EMSA)观察ArgR蛋白与其在体外的相互作用。结果表明,ArgR蛋白能够在体外与argC基因-26～+32 bp、argB基因-86～-29 bp、argG基因-161～-104 bp、carA基因-99～-42 bp结合,且与argB基因的结合程度最大,但不与argJ、argF、argH基因片段结合。本文首次将argC、argB、argG、carA基因中ArgR蛋白结合的靶标位点定位在60bp内,为更深入的研究谷氨酸棒状杆菌ATCC 14067中精氨酸合成的调控机制提供了理论依据。
In bacteria,the repressor protein inhibits the transcription of arginine biosynthesis related genes through binding to conserved sequences in these genes.In Corynebacterium glutamicum ATCC 14067,the genes involved in arginine biosynthesis are located in argCJBDFRGH and carAB gene clusters.The recombinant E.coli BL21(DE3)/pET22 b-argR was constructed to induce the expression of ArgR derived from ATCC 14067 and subsequently achieve the target protein.The analysis of the ArgR protein using molecular sieve columns showed that the relative molecular mass of ArgR protein was about 108 ku,and the ArgR protein was hexamer in the active state.The argC,argJ,argB,argF,argG,argH,carA gene fragments were obtained from biotin-labeled Corynebacterium glutamicum ATCC 14067,and their in vitro interactions with the ArgR protein were examined by electrophoretic mobility shift assays(EMSA).The results showed that the ArgR protein could bind to argC gene at positions-26 ～+32 bp,argB gene at positions-86～-29 bp,argG gene at positions-161～-104 bp,and carA gene at positions-99～-42 bp,and the degree of binding to the argB gene was the greatest whilst no binding between ArgR and the argJ,argF or argH gene fragment.For the first time,the target binding sites of ArgR protein in argC,argB,argG,and carA genes were located within 60 bp,which provided a theoretical basis for further study on the regulatory mechanism of arginine synthesis in Corynebacterium glutamicum ATCC 14067.
In bacteria,the repressor protein inhibits the transcription of arginine biosynthesis related genes through binding to conserved sequences in these genes.In Corynebacterium glutamicum ATCC 14067,the genes involved in arginine biosynthesis are located in argCJBDFRGH and carAB gene clusters.The recombinant E.coli BL21(DE3)/pET22 b-argR was constructed to induce the expression of ArgR derived from ATCC 14067 and subsequently achieve the target protein.The analysis of the ArgR protein using molecular sieve columns showed that the relative molecular mass of ArgR protein was about 108 ku,and the ArgR protein was hexamer in the active state.The argC,argJ,argB,argF,argG,argH,carA gene fragments were obtained from biotin-labeled Corynebacterium glutamicum ATCC 14067,and their in vitro interactions with the ArgR protein were examined by electrophoretic mobility shift assays(EMSA).The results showed that the ArgR protein could bind to argC gene at positions-26 ～+32 bp,argB gene at positions-86～-29 bp,argG gene at positions-161～-104 bp,and carA gene at positions-99～-42 bp,and the degree of binding to the argB gene was the greatest whilst no binding between ArgR and the argJ,argF or argH gene fragment.For the first time,the target binding sites of ArgR protein in argC,argB,argG,and carA genes were located within 60 bp,which provided a theoretical basis for further study on the regulatory mechanism of arginine synthesis in Corynebacterium glutamicum ATCC 14067.
引文
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[11]Xu M J,Rao Z M,Dou W F,et al.The role of ARGRrepressor regulation on L-arginine production in Corynebacterium crenatum[J].Applied Biochemistry and Biotechnology,2013,170(3):587-597
[12]Lee S Y,Park J-M,Lee J H,et al.Interaction of transcriptional repressor Arg R with transcriptional regulator Far R at the arg B Promoter region in Corynebacterium glutamicum[J].Applied and Environmental Microbiology,2011,77(3):711-718
[13]Yim S H,Jung S,Lee S K,et al.Purification and characterization of an arginine regulatory protein,Arg R,in Corynebacterium glutamicum[J].Journal of Industrial Microbiology&Biotechnology,2011,38(12):1911-1920
[14]闫洪波,王威,李令娣,等.原核细胞精氨酸生物合成途径的研究进展[J].生物技术通报,2015,31(1):21-28YAN Hong-bo,WANG Wei,LI Ling-di,et al.Research progress of the arginine biosynthetic pathway in prokaryotic cells[J].Biotechnology Bulletin,2015,31(1):21-28
[15]Iwanaga N,Ide K,Nagashima T,et al.Genome-wide comprehensive analysis of transcriptional regulation by ArgRin Thermus thermophilus[J].Extremophiles,2014,18(6):995-1008
[16]Cho S,Cho Y B,Kang T J,et al.The architecture of Arg R-DNA complexes at the genome-scale in Escherichia coli[J].Nucleic Acids Research,2015,43(6):3079-3088
[17]Perez-Redondo R,Rodriguez-Garcia A,Botas A,et al.ArgRof Streptomyces coelicolor is a versatile regulator[J].Plos One,2012,7(3):16
[2]Theron G,Reid S J.Arg R-promoter interactions in Corynebacterium glutamicum arginine biosynthesis[J].Biotechnology and Applied Biochemistry,2011,58(2):119-127
[3]Lee S Y,Shin H S,Park J S,et al.Proline reduces the binding of transcriptional regulator Arg R to upstream of arg B in Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology,2010,86(1):235-242
[4]袁永.钝齿棒杆菌Arg R的凝胶阻滞实验及单增李斯特菌act A、inl B、p60表达载体的构建[D].南昌:南昌大学,2012YUAN Yong.Electrophoretic mobility shift assay by Arg Rrepressor from Corynebacterium crenatum and construction of expression vector carrying act A,inl B,p60 from Listeria monocytogenes[D].Nanchang:Nanchang University,2012
[5]Lv Y Y,Liao J J,Wu Z H,et al.Genome sequence of Corynebacterium glutamicum ATCC 14067,which provides insight into amino acid biosynthesis in coryneform bacteria[J].Journal of Bacteriology,2012,194(3):742-743
[6]Kalinowski J,Bathe B,Bartels D,et al.The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins[J].Journal of Biotechnology,2003,104(1-3):5-25
[7]Molloy P L.Electrophoretic mobility shift assays[J].Methods in Molecular Biology(Clifton,N.J.),2000,130:235-246
[8]Hellman L M,Fried M G.Electrophoretic mobility shift assay(EMSA)for detecting protein-nucleic acid interactions[J].Nature Protocols,2007,2(8):1849-1861
[9]Charlier D,Roovers M,Van Vliet F,et al.Arginine regulon of Escherichia coli K-12.A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression[J].Journal of Molecular Biology,1992,226(2):367-386
[10]Tian G,Lim D,Carey J,et al.Binding of the arginine repressor of Escherichia coli K12 to its operator sites[J].Journal of Molecular Biology,1992,226(2):387-397
[11]Xu M J,Rao Z M,Dou W F,et al.The role of ARGRrepressor regulation on L-arginine production in Corynebacterium crenatum[J].Applied Biochemistry and Biotechnology,2013,170(3):587-597
[12]Lee S Y,Park J-M,Lee J H,et al.Interaction of transcriptional repressor Arg R with transcriptional regulator Far R at the arg B Promoter region in Corynebacterium glutamicum[J].Applied and Environmental Microbiology,2011,77(3):711-718
[13]Yim S H,Jung S,Lee S K,et al.Purification and characterization of an arginine regulatory protein,Arg R,in Corynebacterium glutamicum[J].Journal of Industrial Microbiology&Biotechnology,2011,38(12):1911-1920
[14]闫洪波,王威,李令娣,等.原核细胞精氨酸生物合成途径的研究进展[J].生物技术通报,2015,31(1):21-28YAN Hong-bo,WANG Wei,LI Ling-di,et al.Research progress of the arginine biosynthetic pathway in prokaryotic cells[J].Biotechnology Bulletin,2015,31(1):21-28
[15]Iwanaga N,Ide K,Nagashima T,et al.Genome-wide comprehensive analysis of transcriptional regulation by ArgRin Thermus thermophilus[J].Extremophiles,2014,18(6):995-1008
[16]Cho S,Cho Y B,Kang T J,et al.The architecture of Arg R-DNA complexes at the genome-scale in Escherichia coli[J].Nucleic Acids Research,2015,43(6):3079-3088
[17]Perez-Redondo R,Rodriguez-Garcia A,Botas A,et al.ArgRof Streptomyces coelicolor is a versatile regulator[J].Plos One,2012,7(3):16