溶磷菌Pseudomonas sp. wj1的Pst系统鉴定及pstS基因功能分析

Identification of the Pst system of phosphate solubilizing bacteria Pseudomonas sp. wj1 and functional analysis of pstS gene

为明确溶磷菌wj1的无机磷酸盐(Pi)的转运机制,对溶磷菌wj1磷酸盐特异性转运系统(Pst)进行生物信息学分析并与土壤中常见细菌的Pst系统进行比较,通过亚克隆构建溶磷菌wj1pstS蛋白的原核表达系统,利用钼酸铵分光光度法测定诱导表达的pstS蛋白对磷酸盐的结合率。结果表明:溶磷菌wj1的Pst系统是由pstS、pstC、pstA、pstB和phoU 5个基因组成,且依次分布于染色体上,这种结构与土壤中常见细菌的大多数菌属相似。溶磷菌wj1的pstS蛋白位于细胞质外,是一种具有信号肽的可溶性蛋白,其空间结构是由2个球状结构域组成。与绿脓假单胞菌的pstS蛋白结构相比,溶磷菌wj1pstS蛋白的活性中心位于两个球状结构域的裂缝中,其中有9个氨基酸参与Pi特异性结合,1个氨基酸通过疏水作用维持蛋白与Pi的结合。溶磷菌wj1pstS蛋白在大肠杆菌中过量表达后,使得重组大肠杆菌的无机磷酸盐吸收率显著提高,表明溶磷菌wj1pstS蛋白具有结合Pi的能力,但菌浓度不变时Pi浓度的增加会抑制溶磷菌wj1pstS蛋白对Pi的结合。

To understand the mechanism of Pi transport by phosphate-solubilizing bacteria（Psb）,the specific phosphate transport system（Pst）of Psb wj1 was investigated by bioinformatics and compared with that of common bacteria in soil.The prokaryotic expression system of strain wj1 pstS protein was constructed,and the binding rate of the induced pstS protein in Escherichia coli to phosphate was determined by ammonium molybdate spectrophotometric method.The results showed that：The Pst system of phosphate solubilizing bacteria wj1 was composed of pstS,pstC,pstA,pstB and phoU,which were sequentially distributed on the chromosome,and the structure of Pst system of most common bacteria in soil was similar;The pstS protein with signal peptide located in the extracellular was consisted of two globular domains;Compared with pstS protein of Pseudomonas aeruginosa,the crack between two globular domains was the active center of the protein,and the amino acid residues involved in Pi specific binding site were found.The phosphate binding rate of recombinant E.coli was dramatically increased,indicating that the phosphate solubilizing bacteria wj1 pstS protein had the ability of binding Pi,and the binding rate of pstS to Pi decreased when Pi concentrationin solution was increased and the concentration of bacteria remained the same.