【作者】 周贤轩；

【导师】 孙宝林；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 在细菌的生活过程中,环境条件、细菌的密度及生理状态常常处于变化之中。经过长期的进化过程,为了对外部环境及胞内生理状态的变化作出及时反应,细菌已经学会利用各种小分子化合物作为信号分子,并发展出各种信号传递机制去捕获及把这些信号传递到细胞中去。细菌细胞间通迅能使细菌在多细胞水平上采取协调一致的行动,这一过程通常又称为群体感应或密度依赖的基因调控,即细菌在增殖的过程中,不断地向胞外分泌信号小分子,这种信号小分子被称为自诱导物。随着细菌数量的增加,自诱导物就在周围环境中逐渐积累,当自诱导物达到一定的阈值浓度时,即可进入细胞,与受体结合,从而开启或关闭一些基因的转录。在大肠杆菌K-12菌株中,存在两套群体感应信号系统,以AI-1为信号分子的群体感应系统一及以AI-2为信号分子的群体感应系统二。群体感应系统一包括与LuxR及LuxI同源的一对蛋白。群体感应系统一的信号分子为AI-1,它由LuxI合成,它的受体蛋白为LuxR。大肠杆菌中的LuxR同源蛋白为SdiA,但无LuxI同源蛋白且不能合成任何AI-1信号分子。但是大肠杆菌的SdiA蛋白能够感应来自其它细菌的AI-1信号分子。最近有报道指出,大肠杆菌通过利用SdiA感应环境中的吲哚及AI-l的浓度变化来控制生物膜的形成,至于SdiA能否直接与吲哚结合,目前仍没有确切的答案。研究表明,SdiA能够促进或抑制核糖核酸多聚酶与启动子的结合,从而控制目的基因的转录。细胞中过量表达SdiA能够影响许多基因的表达,其中包括一些与细胞分裂、运动性、趋化性、及药物代谢相关的基因。大肠杆菌有一个完整的群体感应系统二,共包括九个基因,即luxS、lsrR、IsrK、及lsrACDBFG。cAMP-CRP复合体促进lsrR及lsr操纵子(lsrACDBFG)的表达,lsrR与lsr操纵子相邻但转录方向相反,LsrR能够抑制lsrR及lsr操纵子的表达。由LuxS合成群体感应系统二的信号分子,即AI-2,随着细菌的增殖,该信号分子在胞外不断地积累。当胞外的AI-2达到一定的阈值浓度时,由lsr操纵子编码的载体运入胞内,然后被LsrK磷酸化,磷酸化的AI-2与LsrR结合并改变其构象,从而达到调控目的。LsrF与LsrG参与后续的磷酸化AI-2的降解过程。最近研究表明,lsrR或lsrK突变株形成生物膜的厚度及总量均较野生株有所降低。同时,lsrR或lsrK突变株中的sRNA,即全局调控因子DsrA及细胞分裂抑制因子DicF的表达增加了2—4.4倍。在弧菌Vibrio harveyi中,有三条群体感应信号通路,这三条通路会聚到一起,控制同一套基因的表达,sRNA也参与了这一调控过程。在大肠杆菌中,仅知道cAMP-CRP复合体通过结合到lsrR及lsr操纵子的启动序列上,促进它们的表达,LsrR的作用正好相反。然而,大肠杆菌中群体感应系统一与系统二之间的相互作用关系仍不清楚。为了更全面地了解大肠杆菌群体感应信号途径,及群体感应与其它信号通路之间的关系,本研究采用基因敲除、凝胶阻滞及酶学分析等方法,分析了SdiA对YdiV的转录调控作用,及SdiA与群体感应系统二的相互作用。环二鸟苷酸是细菌中的第二信使,它由含GGDEF结构域的蛋白合成,被含EAL结构域的蛋白降解。YdiV就是一个含EAL结构域的蛋白。在本研究中,我们发现sdiA及ydiV的表达同时受到葡萄糖的抑制。SdiA能够结合到ydiV的启动子区域,来自于其它细菌的AI-1在非sdiA突变株中能够促进ydiV的表达。通过进一步的研究,我们还发现SdiA具有非特异性地结合DNA的能力。在sdiA及ydiV双突变株中,而不是单突变株中,胞内的cAMP浓度降低了2倍,cAMP浓底的降低使大肠杆菌中群体感应系统二受到抑制。这些研究结果说明了YdiV及cAMP介导了大肠杆菌中两个群体感应系统之间的相互作用;并提示了大肠杆菌中感应各种环境因子的信号通路,如自诱导物及葡萄糖相关信号途径,通过一定的方式联系起来,一起控制细菌的系列行为。更多还原

【Abstract】 In the life span of bacteria, environmental cues, density of bacteria and physiological states keep changing. To respond to these variable conditions, bacteria have learned how to use small molecular as signal, and evolved a variety of signal sensing and delivering mechanisms during the long evolution progress. Cell-to-cell communication in bacteria that leads to co-ordinated behaviour at a multicellular level is often referred to as quorum sensing, or density-dependent gene regulation, in this process bacteria produce, release and respond to signaling molecules called autoinducers. While bacteria proliferated, autoinducers accumulated in the around environment, once reached a threshold autoinducers can be detected and enter the cell, bind to its receptors and regulate transcription of lots of target genes. Escherichia coli K-12 has two kinds of quorum sensing systems, system 1 which uses AI-1 as signal molecular, and system 2 which uses AI-2 as signal molecular.Quorum sensing system 1 includes protein pairs similar to LuxR and LuxI. The system 1 autoinducer named AM is produced by LuxI and is detected by LuxR. E. coli encodes a LuxR homologue, SdiA, but it does not encode a LuxI homologue or synthesize any AHL molecule detected by SdiA. However, SdiA of E. coli responds to several AHLs generated by other microbial species. Recently a report indicates that E. coli uses SdiA to monitor indole and AHLs to control biofilms, however, it is not ascertained whether indole itself binds to SdiA. A previous study shows that SdiA can support or inhibit RNA polymerase binding to the promoters and thereby affects transcription of the target genes. Overexpression of SdiA affects expression of a battery of genes, including cell division, motility, chemotaxis, and multidrug efflux pump genes.E. coli has an intact quorum sensing system 2 including nine genes (e.g. luxS, lsrR, lsrK and lsrACDBFG). cAMP-CRP complex stimulates expression of both lsrR and lsr operon which includes IsrACDBFG, while LsrR represses their expression and is located adjacent to but is transcribed divergently from the lsr operon. The system 2 autoinducer named AI-2 is synthesized by LuxS and accumulates extracellularly. Following internalization by the Lsr transporter encoded by the genes in the lsr operon, AI-2 is phosphorylated by LsrK and phospho-AI-2 binds specifically to LsrR and antagonizes it. LsrF and LsrG are required for further processing of phospho-AI-2. It has been recently reported that the mean biofilm thickness and biomass of the lsrR or lsrK mutant are lower than that of the wild type, meanwhile, the global small RNA (sRNA) regulator DsrA and the sRNA cell division inhibitor DicF are induced 2 to 4.4-fold in both lsrR and lsrK mutants.Three quorum sensing circuits of Vibrio harveyi converge to control a same set of genes, in which sRNA species are involved. In E. coli, cAMP-CRP complex stimulates expression of both lsrR and lsr operon by binding to the promoter regions, while LsrR represses their expression. The relationship between QS system 1 and QS system 2 in E. coli, however, remains obscure. To further explore quorum sensing in E. coli, and the relationship between quorum sensing and other signaling pathways, we analyzed the transcription regulation of ydiV expression by SdiA, and the interaction between SdiA and quorum sensing system 2, by employing these biological technologies, such as gene knock out, gel shift and enzyme activity assay, et al. A class of enzymes containing GGDEF domains synthesize the second messenger c-di-GMP in bacterium that is later hydrolyzed by EAL domain proteins. The protein YdiV consists solely of an EAL domain. Here we show that expression of sdiA and ydiV is inhibited by glucose. SdiA binds to ydiV promoter region in a dose-dependent manner, but nonspecifically in the present study, and AI-1 from other species stimulates ydiV expression in an sdiA -dependent manner. Furthermore, we discover that the double sdiA-ydiV mutation but not the single mutations causes a decrease in intracellular cAMP concentration by 2-fold that leads to the inhibition of QS system 2. These results demonstrate that YdiV and cAMP are involved in the interaction between the two QS systems in E. coli, and indicate that signaling pathways which respond to important environmental cues, such as autoinducers and glucose, are linked together for their control in E. coli.更多还原