摘要
【目的】对嗜水气单胞菌群体感应信号分子AI-2进行细胞外生物合成及活性检测。【方法】对Lux S、MtnN-1、MtnN-2蛋白进行氨基酸序列分析、表达及纯化。以S-腺苷同型半胱氨酸(SAH)为底物,利用纯化的Lux S分别与MtnN-1及MtnN-2蛋白共同作用合成AI-2,并利用哈维氏弧菌报告菌株BB170检测AI-2活性。【结果】嗜水气单胞菌培养液上清中AI-2活性在8 h达到空白对照的16.96倍。氨基酸序列分析表明,嗜水气单胞菌与水生病原菌哈维式弧菌和迟钝爱德华氏Lux S一致性达到76%以上,MtnN-1与MtnN-2氨基酸序列一致性为26.37%,其中MtnN-2与哈维氏弧菌和迟钝爱德华氏菌Pfs一致性达到53%以上。成功表达及纯化了Lux S、MtnN-1和MtnN-2蛋白,细胞外Lux S和MtnN-1共同作用合成的AI-2活性是空白对照的45.04倍,Lux S和MtnN-2共同作用合成的AI-2活性是空白对照的63.62倍。【结论】嗜水气单胞菌能够合成信号分子AI-2。MtnN-1和MtnN-2氨基酸序列尽管存在较大差异,但两者均能与Lux S共同催化AI-2的细胞外生物合成。
[Objective] To realize in vitro biosynthesis and activity detection of quorum sensing signal molecule autoinducer 2 （AI-2） of Aeromonas hydrophila. [Methods] LuxS, MtnN-1, MtnN-2 proteins were analyzed for amino acid sequences, expressed and purified. AI-2 was synthesized from S-adenosylhomocysteine （SAH） with purified LuxS and MtnN-1, or purified LuxS and MtnN-2 separately. The activity of AI-2 was detected by the reporter strain Vibrio harveyi BB 170. [Results] The activity ofAI-2 inA. hydrophila culture supematant was 16.96-fold of the control at 8 h. As analysis of amino acid sequence alignment indicated, the identity of LuxS ofA. hydrophila with that of V. harveyi and Edwardsiella tarda is above 76%. The identity of MtnN-1 with MtnN-2 is 26.37%. MtnN-2 has relatively high homologies with representative Pfs amino acid sequences. The identity of MtnN-2 with that of V. harveyi and E. tarda is above 53%. LuxS, MtnN-1 and MtnN-2 proteins were successfully expressed and purified. The activity of AI-2 produced with purified LuxS and MtnN-1 in vitro was 45.04-fold of the control; the activity of AI-2 produced with purified LuxS and MtnN-2 in vitro was 63.62-fold of the control. [Conclusion] A. hydrophila could secrete AI-2. MtnN-1 and MtnN-2 could both cooperate with LuxS and realize in vitro biosynthesis of AI-2, though the amino acid sequences between the two showed relatively big differences.
出处
《微生物学通报》
CAS
CSCD
北大核心
2015年第10期1858-1865,共8页
Microbiology China
基金
国家自然科学基金项目(No.21272031)
辽宁省教育厅一般项目(No.L2013508)
