变形假单胞菌2-酮基葡萄糖酸操纵子的克隆与分析
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摘要
从变形假单胞菌JUIM01中克隆2-酮基葡萄糖酸操纵子(kgu操纵子),明确其基因组成及生物学信息。根据报道的假单胞菌的基因组信息设计简并引物,采用LA-PCR技术克隆变形假单胞菌的kgu操纵子,对克隆的基因片段进行测序,在此基础上利用生物信息学方法进行分析。结果表明:克隆到的基因片段全长为6 130bp,其中包括基因ptx S、kgu E、kgu K、kgu T和kgu D,分别编码2-酮基葡萄糖酸代谢调控蛋白Ptx S、2-酮基葡萄糖酸差向异构酶、2-酮基葡萄糖酸激酶、2-酮基葡萄糖酸转运蛋白和2-酮基葡萄糖酸-6-磷酸还原酶;这些基因与铜绿假单胞菌PAO1的kgu操纵子中相应基因的序列一致性达56.15%~76.74%,然而变形假单胞菌的ptx S很可能并不位于kgu操纵子中。研究首次从变形假单胞菌中克隆到kgu操纵子,并对其进行生物信息学分析,为变形假单胞菌2-酮基葡萄糖酸代谢机理的研究奠定了基础。
To clone 2-ketogluconate utilization operon(kgu operon) from Pseudomonas plecoglossicida JUIM01, and to find its genetic organization and biological information. The primers were designed on the basis of available sequences from Ps. aeruginosa, and kgu operon was amplified by LA-PCR. The cloned fragment was sequenced and analyzed by bioinformatics. The results showed that entire nucleotide sequences(6 130 bp) were involved in the catabolism of 2 KGA and revealed five open reading frames corresponding to the ptx S gene encoding 2-ketogluconate metabolism regulation protein, the kgu E gene encoding 2-ketogluconate epimerase, the kgu K gene encoding 2-ketogluconate kinase, the kgu T gene encoding 2-ketogluconate transport protein and the kgu D gene encoding 2-ketogluconate-6-phosphate reductase.These genes were predicted to share 56.15%~76.74% sequence identity with the corresponding genes from Ps. aeruginosa PAO1. Besides, the ptx S gene might not reside in the kgu operon. The kgu operon was amplified successfully from the Ps. plecoglossicida for the first time and was analyzed by the bioinformatics. The results of this study may provide a foundation for further study about the 2-ketogluconate metabolism in Ps. plecoglossicida.
To clone 2-ketogluconate utilization operon(kgu operon) from Pseudomonas plecoglossicida JUIM01, and to find its genetic organization and biological information. The primers were designed on the basis of available sequences from Ps. aeruginosa, and kgu operon was amplified by LA-PCR. The cloned fragment was sequenced and analyzed by bioinformatics. The results showed that entire nucleotide sequences(6 130 bp) were involved in the catabolism of 2 KGA and revealed five open reading frames corresponding to the ptx S gene encoding 2-ketogluconate metabolism regulation protein, the kgu E gene encoding 2-ketogluconate epimerase, the kgu K gene encoding 2-ketogluconate kinase, the kgu T gene encoding 2-ketogluconate transport protein and the kgu D gene encoding 2-ketogluconate-6-phosphate reductase.These genes were predicted to share 56.15%~76.74% sequence identity with the corresponding genes from Ps. aeruginosa PAO1. Besides, the ptx S gene might not reside in the kgu operon. The kgu operon was amplified successfully from the Ps. plecoglossicida for the first time and was analyzed by the bioinformatics. The results of this study may provide a foundation for further study about the 2-ketogluconate metabolism in Ps. plecoglossicida.
引文
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[3]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.
[4]孙文敬,高培玲,魏转,等.球形节杆菌C224分批发酵2-酮基-D-葡萄糖酸的条件优化及其动力学模型的构建[J].食品与发酵工业,2013,39(5):31-36.
[5]魏转,余泗莲,孙文敬,等.2-酮基-D-葡萄糖酸发酵生产研究进展[J].食品科学,2008,29(8):636-639.
[6]CHIA M,NGUYEN T B V,CHOI W J.DO-stat fed-batch production of 2-keto-D-gluconic acid from cassava using immobilized Pseudomonas aeruginosa[J].Applied Microbiology and Biotechnology,2008,78(5):759-765.
[7]CHOI W J,LEE E Y,CHOI C Y.Effect of dissolved oxygen concentration on the metabolism of glucose in Pseudomonas putida BM014[J].Biotechnology and Bioprocess Engineering,1998,3(2):109-111.
[8]DEL C 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.
[9]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.
[10]SWANSON B L,HAGER P,JR P P,et al.Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1[J].Molecular Microbiology,2000,37(3):561-573.
[11]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.
[12]朱玉贤,李毅.现代分子生物学[M].北京:高等教育出版社,2002:71-74.
[13]徐建中.枯草杆菌rib操纵子结构基因在ccp A位点的表达[D].天津:天津大学,2008.
[14]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.