植物乳杆菌种间群体感应信号分子合成关键基因luxS的功能研究
发布时间：2018-10-15 18:40
【摘要】：群体感应(Quorum Sensing,QS)是细菌细胞之间进行信息交流的一种机制。乳酸菌属于革兰氏阳性细菌,除了拥有寡肽介导的QS系统,还同时拥有自诱导物2(autoinducer 2,AI-2)介导的种间QS系统,研究发现QS系统与乳酸菌耐酸耐胆盐及抑菌特性有较为密切的关系。本研究使用的植物乳杆菌(Lactobacillus plantarum)AY01与YM-4-3分别分离自石林发酵羊奶与易门豆豉,且在对AY01与YM-4-3的全基因组序列进行分析时发现,AY01含有一个编码种间QS系统信号分子——AI-2合成酶(LuxS)的开放阅读框(Open Reading Frame,ORF),而YM-4-3含有两个。本论文主要针对AY01 luxS的敲除及其luxS突变株在酸、胆盐耐受、生物膜形成能力及抑制肠出血性大肠杆菌(enterohemorrhage Escherichia coli,EHEC)生物膜形成等方面与野生型菌株的差异。此研究基础上,构建了两种luxS基因的原核表达载体pET28a-luxS1和pET28a-luxS2,并成功在E.coli BL21(DE3)中表达。具体实验结果如下:(1)利用温度敏感型敲除载体PKML05,通过同源重组方法敲除了 AY01的luxS,得到luxS突变菌株LuxSA1。(2)哈氏弧菌(Vibrio harveyi)BB170的生物发光实验显示LuxSA1不能产生AI-2,表明luxS在AY01合成AI-2途径中发挥关键性作用。(3)AY01与LuxSA1在生长、产酸、酸与胆盐耐受及生物膜形成能力方面没有显著性差异。(4)通过AY01与LuxS△1对Caco-2细胞的粘附实验发现,培养14 h与27 h的LuxSA1菌体的粘附能力远低于AY01,证明luxS在AY01对Caco-2细胞的粘附过程中发挥重要作用。(5)AY01培养物对EHEC生物膜形成的抑制作用强于LuxSA1。(6)构建了两种luxS基因原核表达载体pET28a-luxS1和pET28a-luxS2,并通过优化纯化步骤成功纯化出纯度超过90%的LuxS 1与LuxS2蛋白。(7)纯化的LuxS1与LuxS2都能催化SRH合成QS信号分子AI-2,诱导V.harveyi BB170 生物发光;Al3+,Ca2+和 EDTA 能增强 LuxS1 的酶活性,Al3+,Ca2+,Fe2+,Mn2+和EDTA能增强LuxS2的酶活性,LuxS1与LuxS2都受到Cu2+,Zn2+,Ni2+等种金属离子及甘油的强烈抑制。
[Abstract]:Group sensing (Quorum Sensing,QS) is a mechanism for the communication of information between bacterial cells. Lactic acid bacteria (lactic acid bacteria) belong to Gram-positive bacteria. In addition to the QS system mediated by oligoseptide, lactic acid bacteria also possess the interspecific QS system mediated by self-inducible agent 2 (autoinducer 2 / AI-2). It is found that the QS system is closely related to acid tolerance and bile salt tolerance and bacteriostatic characteristics of lactic acid bacteria. Lactobacillus plantarum (Lactobacillus plantarum) AY01 and YM-4-3 were isolated from fermented goat milk and Yimen Douchi, respectively. By analyzing the whole genome sequence of AY01 and YM-4-3, we found that AY01 contains an open reading frame (Open Reading Frame,ORF of AI-2 synthase (LuxS), and YM-4-3 contains two. This paper aims at the difference between AY01 luxS knockout and luxS mutant in acid, bile salt tolerance, biofilm formation and inhibition of (enterohemorrhage Escherichia coli,EHEC biofilm formation in enterohemorrhagic Escherichia coli. Based on this study, two prokaryotic expression vectors of luxS gene, pET28a-luxS1 and pET28a-luxS2, were constructed and successfully expressed in E.coli BL21 (DE3). The specific results are as follows: (1) the bioluminescence experiments of luxS mutant LuxSA1. (2) (Vibrio harveyi) BB170 showed that LuxSA1 could not produce AI-2, and luxS could not be produced in AY01 by using the thermo-sensitive knockout vector PKML05, to knock out the luxS, of AY01 by homologous recombination method. Synthesis of AI-2 pathway plays a key role. (3) AY01 and LuxSA1 in growth, There was no significant difference in acid production, acid and bile salt tolerance and biofilm formation ability. (4) Adhesion of AY01 and LuxS 1 to Caco-2 cells was observed. The adhesion ability of LuxSA1 cells cultured for 14 h and 27 h was much lower than that of AY01,. It was proved that luxS played an important role in the adhesion of AY01 to Caco-2 cells. (5) the inhibitory effect of AY01 culture on EHEC biofilm formation was stronger than that of LuxSA1. (6). Two luxS gene prokaryotes were constructed. The expression vectors pET28a-luxS1 and pET28a-luxS2, were successfully purified with purity of more than 90% of LuxS 1 and LuxS2 protein. (7) the purified LuxS1 and LuxS2 could catalyze SRH to synthesize QS signaling molecule AI-2, to induce V.harveyi BB170 bioluminescence; Al3, Ca2 and EDTA could enhance the expression of V.harveyi BB170 bioluminescence. The enzyme activity of LuxS1, Al3, Ca2, Fe2, Mn2 and EDTA enhanced the activity of LuxS2. LuxS1 and LuxS2 were strongly inhibited by Cu2, Zn2, Ni2 and other metal ions and glycerol.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
本文编号：2273472
[Abstract]:Group sensing (Quorum Sensing,QS) is a mechanism for the communication of information between bacterial cells. Lactic acid bacteria (lactic acid bacteria) belong to Gram-positive bacteria. In addition to the QS system mediated by oligoseptide, lactic acid bacteria also possess the interspecific QS system mediated by self-inducible agent 2 (autoinducer 2 / AI-2). It is found that the QS system is closely related to acid tolerance and bile salt tolerance and bacteriostatic characteristics of lactic acid bacteria. Lactobacillus plantarum (Lactobacillus plantarum) AY01 and YM-4-3 were isolated from fermented goat milk and Yimen Douchi, respectively. By analyzing the whole genome sequence of AY01 and YM-4-3, we found that AY01 contains an open reading frame (Open Reading Frame,ORF of AI-2 synthase (LuxS), and YM-4-3 contains two. This paper aims at the difference between AY01 luxS knockout and luxS mutant in acid, bile salt tolerance, biofilm formation and inhibition of (enterohemorrhage Escherichia coli,EHEC biofilm formation in enterohemorrhagic Escherichia coli. Based on this study, two prokaryotic expression vectors of luxS gene, pET28a-luxS1 and pET28a-luxS2, were constructed and successfully expressed in E.coli BL21 (DE3). The specific results are as follows: (1) the bioluminescence experiments of luxS mutant LuxSA1. (2) (Vibrio harveyi) BB170 showed that LuxSA1 could not produce AI-2, and luxS could not be produced in AY01 by using the thermo-sensitive knockout vector PKML05, to knock out the luxS, of AY01 by homologous recombination method. Synthesis of AI-2 pathway plays a key role. (3) AY01 and LuxSA1 in growth, There was no significant difference in acid production, acid and bile salt tolerance and biofilm formation ability. (4) Adhesion of AY01 and LuxS 1 to Caco-2 cells was observed. The adhesion ability of LuxSA1 cells cultured for 14 h and 27 h was much lower than that of AY01,. It was proved that luxS played an important role in the adhesion of AY01 to Caco-2 cells. (5) the inhibitory effect of AY01 culture on EHEC biofilm formation was stronger than that of LuxSA1. (6). Two luxS gene prokaryotes were constructed. The expression vectors pET28a-luxS1 and pET28a-luxS2, were successfully purified with purity of more than 90% of LuxS 1 and LuxS2 protein. (7) the purified LuxS1 and LuxS2 could catalyze SRH to synthesize QS signaling molecule AI-2, to induce V.harveyi BB170 bioluminescence; Al3, Ca2 and EDTA could enhance the expression of V.harveyi BB170 bioluminescence. The enzyme activity of LuxS1, Al3, Ca2, Fe2, Mn2 and EDTA enhanced the activity of LuxS2. LuxS1 and LuxS2 were strongly inhibited by Cu2, Zn2, Ni2 and other metal ions and glycerol.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
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