诱导细菌胞外蛋白酶表达的胞外诱导物和细胞膜上的信号感应受体研究

Secreting extracellular protease by sensing the existence of extracellular proteins is a basic physiological reaction for microorganisms growing with proteins as the sole nitrogen source. However, the mechanism that a strain expresses and secretes its extracellular protease by sensing the existence of extracellular proteins is still unclear. Pseudoalteromonas sp. SM9913 is a bacterial strain with high extracellular protease production. Protease MCP-01 is the main extracellular protease of SM9913. Previous study indicated that the peptide transporter Dtp on the cell membrane of SM9913 may be the receptor for MCP-01 expression. In this programm, the peptides as an inducer and the function and mechanism of Dtp as a transceptor for MCP-01 expression will be further studied. Firstly, the type and concentration of peptides as an inducer for MCP-01 expression will be studied by using various oligo-peptides derived from casein degradation by MCP-01 to induce MCP-01 gene expression. Secondly, the function of Dtp as a tranceptor for MCP-01 expression, as well as its structural basis and molecular mechanism will be analyzed. Finally, the generality of Dtp as a tranceptor for bacterial extracellular protease expression will be analyzed. The results of this study will provide important evidence for the complete reveal of the mechanism of the inducible expression of bacterial extracellular protease.

通过感知胞外蛋白质的存在诱导胞外蛋白酶的表达和分泌是微生物在以蛋白质为氮源的环境中生长的一个基本生理反应，对其生长繁殖非常重要，但微生物如何感知胞外蛋白质的存在并诱导表达胞外蛋白酶却始终没有阐明。Pseudoalteromonas sp. SM9913是一株高产蛋白酶的细菌，主要分泌蛋白酶MCP-01。前期研究表明，该菌株细胞膜上的肽转运蛋白Dtp可能是菌株诱导MCP-01表达时感知胞外诱导物的受体。本项目将进一步研究该菌诱导胞外蛋白酶MCP-01表达的胞外诱导物种类和膜上的信号受体的功能与机制。首先通过分析各种寡肽诱导MCP-01表达情况确定起诱导作用的肽的种类和浓度，然后分析Dtp作为感应和传递诱导肽信号的类转运蛋白受体的功能、结构基础和分子机制。最后分析Dtp作为类转运蛋白受体在细菌中是否具有普遍性。研究结果将为最终揭示微生物诱导胞外蛋白酶表达的机制提供重要依据，具有重要理论意义。

异养细菌分泌的胞外蛋白酶对细菌获取胞外蛋白质氮源非常重要。很多细菌胞外蛋白酶都是诱导型酶，只有当细菌感知到环境中有蛋白质而没有其它更好的氮源时，细菌才分泌胞外蛋白酶将环境中的蛋白质降解为可吸收的氨基酸和寡肽。但是，细菌如何感知胞外蛋白质的存在，并将胞外蛋白质存在的信号传递到胞内激活蛋白酶的表达还很不清楚。本项目以产蛋白酶深海细菌Pseudoalteromonas sp. SM9913 为材料研究其在诱导表达胞外蛋白酶MCP-01过程中感知胞外诱导物并向胞内传递信号的机制，本项目取得如下研究成果：（1）确定了4种三肽是Ps. sp. SM9913 胞外蛋白酶MCP-01 表达的胞外诱导物，其诱导浓度为2 mM。（2）阐明了Ps. sp. SM9913的二三肽转运系统Dtp在MCP-01基因转录表达过程中的作用，其功能是将胞外三肽诱导物转运至胞内。（3）阐明Dtp中转运三肽的关键氨基酸残基，揭示了二三肽转运系统Dtp结合转运三肽向胞内传递信号的分子机制。（4）确定了Ps. sp. CF6-2的胞外蛋白酶Pseudoalterin表达的诱导物是甘氨酸和甘氨酸寡肽, 甘氨酸的有效诱导浓度在0.25 mM-20 mM，并阐明了蛋白酶Pseudoalterin的分泌途径。（5）综合分析了微生物胞外蛋白酶表达的调控机制。本项目已资助12 篇论文发表在国内外微生物学和生物化学领域的主流杂志上。