【作者】 冯德芹；

【导师】 杨苏声；

【作者基本信息】 中国农业大学 ， 微生物学， 2005， 博士

【摘要】 达坂喜盐芽孢杆菌(Halobacillus dabanensis)D-8~T是一株分离自新疆达坂湖的革兰氏阳性中度嗜盐菌,能在0.5%～23%盐浓度的SW培养基上生长,最适生长盐浓度是3%～15%。为揭示革兰氏阳性中度嗜盐菌的耐盐机制,利用差异蛋白质组学研究技术,得到了高分辨率的双向电泳图谱。在电泳胶上清晰地分离出大约700个蛋白点,分子量为17.5～66kDa,等电点范围为3.5～5.9,表明D-8~T菌株中的蛋白质大多数属于酸性蛋白质。此外,大量的高丰度蛋白质集中在4.5～5.0的极窄范围的等电点区域,大多数的低丰度蛋白质分布在图谱的其他部分。 利用蛋白质组学技术研究了D-8~T菌株受到剧烈的高盐冲击时的差异表达蛋白谱,鉴定了一些与高盐冲击相关的蛋白质。采用双向电泳分离技术和ImageMasterTM 2D Platinum图像软件分析方法,对在1%盐浓度下生长的D-8~T菌株(培养18h)和受到25%盐浓度的瞬间冲击5min和50min后的蛋白质表达谱进行比较研究的结果表明,高盐冲击后总计有74个蛋白点表达量显著改变,其中24个蛋白点在高盐下表达量明显增加,8个高盐诱导的新蛋白点,以及42个表达量显著下降的蛋白点.利用MALDI-TOF/MS检测肽质指纹谱和MASCOT检索蛋白质数据库,初步鉴定了7个高盐冲击后表达量增加的蛋白质,它们涉及信号传导途径(类CheY接受器)、能量代谢途径(草酰乙酸脱羧酶、烯醇酶)、热激蛋白和分子伴侣(Ⅰ类热激蛋白,有伴侣活性的ATP酶以及ClpC ATP酶)和抗逆蛋白(烯醇酶)等。此外,ClpC ATP酶是首次鉴定存在于革兰氏阳性中度嗜盐菌中。 为了探讨D-8~T菌株受到低渗沖击时的差异表达蛋白,对生长在20%盐度下的D-8~T曲株(培养18h)受到剧烈的低渗冲击(0%盐度)5min和50min后的蛋白质表达谱进行了比较分析。结果显示,总计有39个蛋白点的表达量发生了明显改变,其中21个蛋白点的表达量明显增加(包括低渗冲击诱导出现的6个新蛋白点),18个蛋白点表达量受到抑制。经过质谱分析和数据库搜索,初步鉴定了5个低渗冲击后表达量增加的蛋白质,分别为形态检测蛋白、青霉素结合蛋白、DnaK、ClpC ATP酶和EPSPS等。此外,热激蛋白是首次鉴定在细菌受到低渗冲击时表达量增加的蛋白质。 研究了D-8~T菌株在不同盐浓度下的蛋白质表达谱,发现133个蛋白点的表达量发生了明显改变。88个蛋白点在10%和20%的高盐度下表达量显著增加,包括26个高盐诱导出现的新蛋白点;45个蛋白点在高盐度下的表达量明显下降,其中包括20个表达消失的蛋白点(有16个蛋白点只是在1%盐度中生长的细胞中才表达)。从质谱分析和蛋白质数据库匹配结果来看,D-8~T菌株在高盐浓度下生长时,表达量明显增加的蛋白质主要有以下几类:与能量代谢相关的蛋白质、参与细胞生存和增殖的蛋白质、作为全局胁迫调节子适应多种胁迫条件的蛋白质、与亲和性溶质如海藻糖和甜菜碱可能相关的蛋白质等。更多还原

【Abstract】 Halobacillus dabanensis D-8~T, a Gram-positive moderately halophilic bacterium, was isolated from the saline deposits of Daban lake in the Xinjiang Uygur Autonomous Region of China. It can grow in the complex medium containing 0.5% to 23% salt with the optimum growth salinity ranging from 3% to 15%. To investigate the salt tolerant mechanisms of the Gram-positive moderately halophilic bacterium D-8~T, the differential proteome techniques was employed and the high resolution 2-D PAGE maps were obtained. About 700 protein spots with the molecular weight between 17.5~66kDa were visualized in the gels, which were spread in the gel of the isoelectric point 3.5 to 5.9. Plenty of high abundance proteins were mainly clustered in the narrow region with the isoelectric point between 4.5 and 5.0, but most of low abundance proteins were in the other region dispersedly. These indicate that the most proteins in the strain D-8~T would be acidic.Some proteins related to salt shock were identified by applying the proteome techniques to analyze the differential protein expression files of the strain D-8~T exposed to severe salt shock. Comparative analysis of the protein expression files was carried out between the strain incubated in the 1% salinity medium (18h) and the ones shocked immediately with high salinity (25%) for 5min and 50min, respectively. There were 74 protein spots expressed differently. Among them, the expression of 24 protein spots were increased markedly while 8 protein spots were induced by salt stress, and the other 42 protein spots were decreased obviously. After hyperosmotic challenge, 7 up-regulated protein spots were identified through MALDI-TOF/MS and MASCOT. They were involved in CheY-like receiver in signal transduction pathways, oxaloacetate decarboxylase and enolase in energy metabolism pathways, heat shock protein and molecular chaperone such as class I heat shock protein, ATPases with chaperone activity, ClpC ATPase et al, and anti-adversity protein such as enolase. It was the first time that ClpC ATPase was identified in Gram-positive moderately halophilic bacteria.To study differential expression protein files of moderately halophilic bacteria subjected to hypoosmotic shock, comparative analysis of the protein expression files was carried out between the strain incubated in the 20% salinity medium (18h) and the ones shocked immediately with low salinity (0%) for 5min and 50min, respectively. There were 39 protein spots expressed differently. Among them, the expression of 21 protein spots were up-regulated clearly involving 6 new induced protein spots and the other 18 protein spots were decreased obviously. After hypoosmotic stress, 5 up-regulated protein spots were identified through MALDI-TOF/MS and MASCOT. They were similar to chaperone protein DnaK, rod shape-determining protein, 5-enolpyruvoylshikimate- 3-phosphate synthase, penicillin-binding protein and ClpC ATPase, respectively. It was the first time that heat shock proteins were found to be induced in bacterium after a shift to low-osmolarity.By means of differential proteomic approach, differential expression protein files of moderately halophilic bacteria grew in different salinity medium was studied, and some proteins related to high salinity were identified. It was found that 133 protein spots expressed differently. In comparison with the sample in 1% salinity, the expression of 88 protein spots was up-regulated remarkably when the strain D-8~T responded to long-term high salt stress between 10% and 20%. Among them, 26 new protein spots were induced by high salt. In addition, 45 protein spots were depressed evidently, among which 20 protein spots disappeared in high salinity. From the results of mass spectrum analyzing and protein databases matching, when the strain D-8~T grew in high salt overexpressed proteins were shown as follows: related to energy-producing pathway, involved in the cell growth and propagation, response to many kinds of stress like global stress regulators and concerned with compatible solutes including trehalose and beta更多还原