变形假单胞菌PtxS蛋白基因的克隆与分析
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摘要
为从变形假单胞菌中克隆2-酮基葡萄糖酸代谢调控蛋白Ptx S的基因,并明确其基本的生物学信息,采用同源比对的方法设计引物,利用TD-PCR技术克隆Ptx S的基因,再用生物信息学方法对其进行分析。试验结果表明:克隆的基因片段全长1 023 bp,其序列与铜绿假单胞菌、恶臭假单胞菌等假单胞菌的转录调控蛋白Ptx S的基因序列一致性达80%左右,编码由340个氨基酸残基组成的蛋白Ptx S。该蛋白与恶臭假单胞菌Ptx S的同源性最高,氨基酸序列一致性达88%。该蛋白定位于细胞质,无信号肽和跨膜区,为亲水性蛋白。该蛋白的氨基酸序列中含有多个结构域,其二级结构中α螺旋、延伸链、β转角和无规卷曲所占的比例分别为54.41%,13.53%,10.88%和21.18%。本研究为该基因的表达及表达产物的分离纯化提供了理论基础。
To clone the gene encoding 2-ketogluconate metabolic regulatory protein Ptx S from Pseudomonas plecoglossicida JUIM01, and to investigate its biological information. The primers were designed by the homologous comparison, and the Ptx S gene was amplified by TD-PCR. The gene was analyzed and the structures and functions of the Ptx S were predicted by the bioinformatic methods. The gene sequence of ptx S revealed a complete open reading frame(ORF) of 1023 bp encoding 340 amino acid residues, and shared about 80% sequence identity with the gene of transcription regulatory protein Ptx S from other pseudomonads such as Ps. aeruginosa, Ps. putida etc. The protein was predicted to be very high homologous to the Ptx S from Ps. putida and share more than 80% sequence identity. This protein was located in the cytoplasm, without transmembrane domain and signal peptide, and was a hydrophilic protein. This protein had some domains and the predicted secondary structure contained 54.41% of α helixes, 13.53% of extended strand, 10.88% of β turns and 21.18% of random coil. The results of this study may provide a stable foundation for the expression and purification of Ptx S protein.
To clone the gene encoding 2-ketogluconate metabolic regulatory protein Ptx S from Pseudomonas plecoglossicida JUIM01, and to investigate its biological information. The primers were designed by the homologous comparison, and the Ptx S gene was amplified by TD-PCR. The gene was analyzed and the structures and functions of the Ptx S were predicted by the bioinformatic methods. The gene sequence of ptx S revealed a complete open reading frame(ORF) of 1023 bp encoding 340 amino acid residues, and shared about 80% sequence identity with the gene of transcription regulatory protein Ptx S from other pseudomonads such as Ps. aeruginosa, Ps. putida etc. The protein was predicted to be very high homologous to the Ptx S from Ps. putida and share more than 80% sequence identity. This protein was located in the cytoplasm, without transmembrane domain and signal peptide, and was a hydrophilic protein. This protein had some domains and the predicted secondary structure contained 54.41% of α helixes, 13.53% of extended strand, 10.88% of β turns and 21.18% of random coil. The results of this study may provide a stable foundation for the expression and purification of Ptx S protein.
引文
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[18]DADDAOUA A,FILLET S,FERN魣NDEZ M,et al.Genes for carbon metabolism and the Tox A 1.75 virulence factor in Pseudomonas aeruginosa are regulated through molecular interactions of Ptx R and Ptx S[J].Plo S One,2012,7(7):e39390.
[19]PINEDA-MOLINA E,DADDAOUA A,KRELL T,et al.In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida Ptx S in complex with DNA[J].Acta Crystallographica Section F-Structural Biology and Crystallization Communications,2012,68(11):1307-1310.
[2]STOTTMEISTER U,AURICH A,WILDE H,et al.White biotechnology for green chemistry:fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses[J].Journal of Industrial Microbiology and Biotechnology,2005,32(11/12):651-664.
[3]XUE Q,WEI Z,SUN W J,et al.2-Keto-D-gluconate-yielding membrane-bound D-glucose dehydrogenase from Arthrobacter globiformis C224:purification and characterization[J].Molecules,2015,20(1):846-862.
[4]YANG G F,WEI Z,SUN W J,et al.Purification and enzymatic characterization of membrane-bound D-gluconate dehydrogenase from Arthrobacter globiformis[J].Journal of Molecular Catalysis B:Enzymatic,2015,113:14-22.
[5]SUN W J,ZHOU Y Z,ZHOU Q,et al.Semi-continuous production of 2-keto-gluconic acid by Pseudomonas fluorescens AR4 from rice starch hydrolysate[J].Bioresource Technology,2012,110:546-551.
[6]TENG W H,SUN W J,YU B,et al.Continuous conversion of rice starch hydrolysate to 2-keto-D-gluconic acid by Arthrobacter globiformis C224[J].Biotechnology and Bioprocess Engineering,2013,18(4):709-714.
[7]HWANGBO H,PARK R D,YONG W K,et al.2-Ketogluconic acid production and phosphate solubilization by Enterobacter intermedium[J].Current Microbiology,2003,47(2):87-92.
[8]WEENK G,OLIJVE W,HARDER W.Ketogluconate formation by Gluconobacter species[J].Applied Microbiology and Biotechnology,1984,20(6):400-405.
[9]SUN W J,YUN Q Q,ZHOU Y Z,et al.Continuous 2-keto-gluconic acid(2KGA)production from corn starch hydrolysate by Pseudomonas fluorescens AR4[J].Biochemical Engineering Journal,2013,77:97-102.
[10]MISENHEIMER T J,ANDERSON R F,LAGODA A A,et al.Production of 2-ketogluconic acid by Serratia marcescens[J].Applied Microbiology,1965,13(3):393-396.
[11]譒VITEL J,譒TURDIK E.2-Ketogluconic acid production by Acetobacter pasteurianu s[J].Applied Biochemistry and Biotechnology,1995,53(1):53-63.
[12]DEL CASTILLO T,RAMOS J L,RODR魱GUEZ-HERVA J J,et al.Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida:genomic and flux analysis[J].Journal of Bacteriology,2007,189(14):5142-5152.
[13]SWANSON B L,HAMOND A N.Autoregulation of the Pseudomonas aeruginosa protein Ptx S occurs through a specific operator site within the ptx S upstream region[J].Journal of Bacteriology,2000,182(15):4366-4371.
[14]SWANSON B L,HAGER P,PHIBBS J R P,et al.Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1[J].Molecular Microbiology,2000,37(3):561-573.
[15]DADDAOUA A,KRELL T,ALFONSO C,et al.Compartmentalized glucose metabolism in Pseudomonas putida is controlled by the Ptx S repressor[J].Journal of Bacteriology,2010,192(17):4357-4366.
[16]WEICKERT M J,ADHYA S.A family of bacterial regulators homologous to Gal and Lac repressors[J].Journal of Biological Chemistry,1992,267(22):15869-15874.
[17]BOUCHEZ D,TOURNEUR J.Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes[J].Plasmid,1991,25(1):27-39.
[18]DADDAOUA A,FILLET S,FERN魣NDEZ M,et al.Genes for carbon metabolism and the Tox A 1.75 virulence factor in Pseudomonas aeruginosa are regulated through molecular interactions of Ptx R and Ptx S[J].Plo S One,2012,7(7):e39390.
[19]PINEDA-MOLINA E,DADDAOUA A,KRELL T,et al.In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida Ptx S in complex with DNA[J].Acta Crystallographica Section F-Structural Biology and Crystallization Communications,2012,68(11):1307-1310.