达坂喜盐芽孢杆菌D-8^T在低渗冲击下的双向凝胶电泳分析

Two-dimensional gel electrophoresis analysis of moderately halophilic bacterium Halobacillus dabanensis D-8^T under hypoosmotic shock condition

为了解革兰氏阳性中度嗜盐菌适应低渗冲击的机制，采用双向凝胶电泳技术，研究达坂喜盐芽孢杆菌（Halobacillus dabanensis）D-8^T在低渗冲击下的差异蛋白表达谱。利用Imagemaster^TM 2D Platinum软件分析到大约650个蛋白点，大多数蛋白分子量分布在17．5～66kDa，等电点为4．0～5．9，偏酸性。在20％盐浓度中生长的D-8菌株受到0％盐浓度的低渗冲击5min及50min后34个蛋白点的表达发生改变，包括20个表达上调的点和14个点表达下调。用MALDI—TOF／MS及MASCOT软件鉴定了4个与低渗胁迫有关的蛋白，分别为热激蛋白DanK、柱状决定蛋白、青霉素结合蛋白和5-莽草酸烯醇式丙酮酸-3-磷酸合成酶。其中，热激蛋白适应低渗胁迫时表达上调为首次报道。

Halobacillus dabanensis D-8^T was isolated from the saline deposits of Daban lake in Xinjiang of China, and is able to grow in complex medium containing 0.5 % to 25 % salt. To figure out the survival mechanisms of Gram-positive moderately halophilic bacteria under hypoosmotic shock conditions, two-dimensional gel electrophoresis （2-DE） was carried out to investigate differential protein expression profiles of H. dabanensis D-8^T in response to low osmotic challenge. The 2-D gels were stored as dry gels and their images were taken by ImageScanner and analyzed by ImagemasterTM 2D Platinum software. About 650 protein spots were detected in 2-D gel. Most of proteins were distributed in molecular mass of 17.5 - 66kDa and the range of isoelectric point 4.0 - 5.9. A total of 34 protein spots were found to alter their expression after strain D-8T was subjected to hypoosmotic shock from 20% to 0% salinity for 5 min and 50 min. Among them, the expression of 20 protein spots is up-regulated including 6 new protein spots, while that of 14 protein spots is down-regulated in answer to sudden osmotic down-shift. Protein spots of interest were excised from the gels and digested by trypsin. By means of matrix-assisted laser desorption ionization-time of flight mass spectrometry （MALDI-TOF/MS） and MASCOT search engine, 4 up-regulated protein spots were identified with peptide mass fingerprint, and are similar to heat shock protein DanK, rod shape-determining protein, penicillin-binding protein （ PBP- 1A） and 5-enolpyruvoylshikimate-3-phosphate synthase, respectively. Noticeably, PBP-1A firstly was up-regulated after shock of 5 min but disappeared after shock of 50 min. This indicated that the strain activate a minor mechanism of peptidoglycan synthesis to compensate the major synthesis mechanism for ceils survival through a down-shift challenge. In addition, this paper was the first report that heat shock proteins were up-regulated in response to sudden osmotic downshift.