群体感应文献

合集下载

1细菌的群体感应

２世纪７年代以后，００研究发现细菌都能合成并释放一种被称为自诱导物质（ｕｏｎｕｅ，Ｉ的信号分子，ａｔｉｄｃｒＡ）当胞外的Ａ浓度达到一个临界浓度时细菌Ｉ问就会通过信号调整行为以适应外界环境，生物发光、抗生素合成等，一如这现象称为群体感应（ｕｒｍｓｎｉｇＱ）。随着对细菌群体感应研究的ｑｏｕｅｓｎ，Ｓ不断深入，人们根据细菌自诱导物的种类，细菌群体感应大致分为三大类：将革兰氏阴性细菌、革兰氏阳性细菌、细菌种间的群体感应。１革兰氏阴性细菌的群体感应１１ＡＬ．Ｈｓ的合成机制革兰氏阴性细菌群体感应的信号分子大多数是Ｎ一乙酰高丝氨酸内酯（Ｈｓ，ＨｓＡＬ）ＡＬ由一个疏水性的保守高丝氨酸内酯环的头部和一个亲水性的可变的酰胺侧链的尾部组成。ＡＬ是以Ｓ腺苷甲硫氨酸（Ａ）酰基化酰基Ｈｓ一ＳＭ和载体蛋白（ｃｌＡＰ为底物，过Ｌｘ蛋白或其类似物将ａｙ— Ｃ的酰基ａｙ— Ｃ）通ｕＩｃｌＡＰ侧链和ＳＭＡ的高半胱氨酸部分的氨基相偶联，再通过酰化作用和内酯化作用脱去酰基载体蛋白（Ｃ —Ｈ并释放出特异的ａｙ — Ｓ。ａｙ— ＣＡＰＳ）ｃｌＨＬｃｌＡＰ中不同的脂肪酰基决定了合成的ＡＬ产物不同，胺链中的碳原子数和第３位上取代基Ｈｓ酰团以及侧链有无一个或多个不饱和键决定了ＡＬＨｓ多样性。１２费氏弧菌（ｉｒｏｆｃｅｉ的群体感应．Ｖｂｊｉｈｒ）ｓ群体感应系统首先是在海洋细菌费氏弧菌（．ｆｓｈｒ）ｖｉｃｅｉ中发现的。ｖ．ｆｓｈｒ的群体感应是Ｎａｓｎｉｃｅｉｅｌｏ等在１７年首次报道了该菌菌体密度与９０生物发光呈正相关，该发光现象受细菌本身的群体感应调节系统所控制。ｖ．ｆｓｈｒ的群体感应是由Ｌｘ和ＬｘｉｃｅｉｕＩｕＲ蛋白调控，ｕＩＬｘ是信号分子ＯＨ（一ＨＬ３

群体感应抑制剂控制微生物污染的研究进展

群体感应抑制剂控制微生物污染的研究进展近年来，微生物污染在医疗、食品、饮用水等领域成为一个备受关注的问题，同时也引起了严重的卫生和经济问题。

传统方法常使用化学药剂对微生物进行控制和消除，但随着对环境保护意识的提高，该方法的应用范围越来越受到限制。

而群体感应抑制剂的出现，为控制微生物污染提供了新思路。

本文通过综述国内外有关群体感应抑制剂控制微生物污染的研究进展，以期为相关学科的研究提供借鉴和参考。

一、群体感应抑制剂的定义和作用机制群体感应抑制剂是一类能够抑制微生物群体感应的物质。

群体感应是微生物细胞间的一种细胞信号传递系统，具有在同一群体内调节基因表达、控制生长和代谢等生理功能的作用。

而群体感应抑制剂则可以干扰这种信号传递系统的正常运作，从而抑制微生物的群体感应和生长。

群体感应抑制剂可以通过多种途径干扰微生物的群体感应系统，例如：（1）光化学物质——例如紫外线、光敏剂等；（2）植物提取物——例如咖啡因、香草酸等；（3）海洋生物——例如藻类、海绵体等；（4）化合物合成——例如多肽、二元素等。

通过上述途径干扰微生物的群体感应系统，可以达到控制微生物生长和繁殖的目的，从而实现对微生物污染的控制。

二、群体感应抑制剂在医疗领域的应用在医疗领域，微生物的感染容易导致严重的健康问题。

传统的抗生素治疗方法存在多种局限性，例如抗生素对特定微生物的敏感性、多重耐药等问题。

群体感应抑制剂作为一种新的治疗方法，可以提供一种替代性的治疗方案。

目前已有多种群体感应抑制剂被应用于医疗领域。

1、肽类群体感应抑制剂肽类群体感应抑制剂是一种与肽类抗生素相近的化合物，具有广谱的抑菌作用。

例如已有报道表明，培养基中添加巴西牛樟脑（HD-034）、庆大霉素类似物（NSTA-4）等肽类群体感应抑制剂，可以抑制病原性菌种的生长、繁殖和生产外毒素等。

2、天然产物群体感应抑制剂天然产物群体感应抑制剂是利用植物、动物等自然界的资源，通过提取和化学合成等方法获得的有效成分。

细菌群体感应系统蛋白质及生理功能发生改变的研究进展

细菌群体感应系统蛋白质及生理功能发生改变的研究进展目录一、内容综述 (2)1. 细菌群体感应系统的概述 (3)2. 研究背景与意义 (4)二、细菌群体感应系统基本原理 (6)1. 群体感应系统的定义 (7)2. 信号分子的种类与作用 (8)3. 信号传递机制 (9)三、细菌群体感应系统蛋白质研究进展 (10)1. 蛋白质的组成与结构 (12)主要蛋白质的发现与功能 (13)蛋白质相互作用网络 (14)2. 蛋白质功能的改变 (15)突变对蛋白质功能的影响 (16)翻译后修饰对蛋白质功能的影响 (17)四、细菌群体感应系统生理功能改变的研究进展 (18)1. 生理功能的改变 (19)对细菌生长、代谢的影响 (20)对细菌毒力与耐药性的影响 (22)2. 与疾病的关系 (22)群体感应系统与感染过程的关系 (24)群体感应系统与抗生素治疗的关系 (25)五、展望与挑战 (27)1. 进一步研究方向 (28)新蛋白质的发现与功能研究 (30)信号传递机制的深入研究 (31)2. 应用前景 (32)抗菌药物的合理设计与开发 (33)细菌疫苗的研发 (35)六、结论 (36)1. 细菌群体感应系统蛋白质及生理功能发生改变的研究成果总结372. 对未来研究的启示与建议 (38)一、内容综述随着分子生物学技术的飞速发展，细菌群体感应系统（Quorum Sensing, QS）的研究取得了显著的进展。

群体感应是指细菌通过分泌和接收信号分子进行信息交流，从而协调群体行为的现象。

在这一系统中，蛋白质扮演着至关重要的角色，其结构和功能的变化直接影响着整个群体的行为和生理状态。

在细菌群体感应系统中，最为经典的两种信号传导方式分别是AI1型和AI2型。

AI1型信号主要依赖于LuxSAI1通路，而AI2型信号则通过LsrABCepA通路进行传输。

这些信号分子的合成和感知主要依赖于特定的蛋白质，如LuxS、LsrB等。

当信号分子浓度达到一定程度时，它们可以启动一系列的基因表达调控，进而影响细菌的生长、代谢、毒力等多个方面。

乳酸菌群体感应的研究进展

2021年第40卷第2期总第348期• 5 •中国酿造专论与综述乳酸菌群体感应的研究进展李雷兵，朱寒剑，郑心，李琴，穆杨，徐宁，胡勇2吴茜,柳志杰，李玮，汪超，周梦J *收稿日期:2020-06-11修回日期：2020-08-01基金项目：国家自然科学基金(31601455)；湖北省粮食局科技创新项目(鄂财商发[2017]58号)；湖北工业大学博士启动基金项目(BSQD14021) 作者简介:李雷兵(1995-),男，硕士研究生，研究方向为食品微生物。

*通讯作者:周梦舟(1986-),男,副教授，博士，研究方向为食品营养安全、食品微生物。

(湖北工业大学工业发酵湖北省协同创新中心湖北省食品发酵工程技术研究中心9湖北武汉430068)摘要:乳酸菌的益生特性已引起公众的广泛关注。

群体感应是细菌感受外界环境变化并做出反应的转导机制，对乳酸菌的存活及益生特性至重要。

因此，近些年来乳酸菌的群体感应研究热点文综述了乳酸菌群体感应的信分子及其分系统，群体感应对乳酸菌环境适应的调控(生物膜、耐酸、耐胆盐)，群体感应对乳酸菌益生特性(抑制致病菌、与宿主 J 的影响以及实际应用，乳酸菌群体感应今的基础研究和工业化应用提参考。

关键词:乳酸菌;群体感应;调控机制中图分类号:TS201.3文章编号：0254-5071 (2021)02-0005-07doi:10.11882/j.issn.0254-5071.2021.02.002引文格式:李雷兵,朱寒剑，郑心，等•乳酸菌群体感应的研究进展!J].中国酿造,2021,40(2):5-11.Research progress of lactic acid bacteria quorum sensingLI Leibing, ZHU Hanjian, ZHENG Xin, LI Qin, MU Yang, XU Ning, HU Yong, WU Qian, LIU Zhijiie,LI Wei, WANG Chao, ZHOU Mengzhou *(Hubei Food Fermentation Engineering Technology Research Center, Hubei Collaborative Innovation Center of I ndustrial Fermentation,Hubei University of Technology, Wuhan 430068, China)Abstract ： The probiotic characteristics of lactic acid bacteria have aroused widespread public concern. Quorum sensing is a transduction mechanism iorbacteria to sense and respond to changes in the external environment, which is very important for the survival and probiotic characteristics of lactic acid bacteria. Therefore, the quorum sensing of lactic acid bacteria has become a research hotspot in recent years. In this paper, the signal molecules andtwo-component systems of quorum sensing of lactic acid bacteria, the regulation of quorum sensing on environmental adaptation of lactic acid bacteria(biofilm, acid tolerance, bile salt tolerance), and the effect of quorum sensing on the probiotic characteristics of lactic acid bacteria (inhibition of pathogenic bacteria, interaction with host) and its practical application were reviewed, in order to provide reference for the basic research and industrial application ofquorum sensing of lactic acid bacteria in the future.Key words ： lactic acid bacteria; quorum sensing; regulation mechanism乳酸菌是我国传统发酵食品中的重要微生物,除了可以提高食品的质量和营养外，还可通过多种机制对人体产生有益影响冋。

e42-1群体感应

e42-1 群体感应
群体感应是指某个菌体能够感应到周围环境中同种细菌的其他成员的存在并做出反应的现象。

在上个世纪60年代后期，J.Woodland Hastings等人发现，某些海洋发光细菌只有在达到临界数量后才会发光，而在细菌数量不足时就保持黯淡。

对此他们认为，细菌释放了一种叫自诱导物（autoinducer）的信号分子，来对生物荧光进行调控，同时用它来监测同种细菌的密度。

直到1981年，他们才首次纯化并确定自诱导物是一种脂酰高丝氨酰内酯（acylated homoserine lactone，AHL）。

目前已知具有群体效应的菌体会持续地释放出自诱导物，随着群体扩展，更多自诱导物被增殖的细菌制造，并释放到菌体周围，其浓度也因此渐渐上升。

一旦自诱导物浓度达到一个临界值，细菌便可感应到群体数目的变化，一些细胞行为也会因此改变，如生物荧光、接合作用、转化作用、孢子生成、生物薄膜（biofilm）形成、抗生素和毒素的合成。

迄今为止，具有群体感应的菌种已达数十种，其中，革兰氏阴性菌有两类自诱导物——AHL和呋喃糖硼酸二酯（furanosyl borate diester），革兰氏阳性菌则以寡肽为自诱导物。

以AHL为自诱导物的系统是由luxI和luxR两个结构基因组成，分别编码AHL合酶和AHL反应调节蛋白。

产生的自诱导物分子需要和反应调节蛋白结合，才能控制与群体感应有关的多个基因的表达（图e42-1），例如Vibrio fischeri及V. harveyi的生物荧光基因。

图e42-1 细菌群体感应的LuxI/LuxR系统。

细菌群体感应及其在病原菌防治中的应用

细菌群体感应及其在病原菌防治中的应用梁心琰;阮海华【摘要】Bacteria releases one or several chemical molecules served as signal to estimate the density of bacteria and sense the change of environment. This chemical communication, called as “quorum sensing”(QS)is defined as a density dependent mechanism by which bacteria coordinate expression of specific target genes in response to a critical concentration of signal molecules. A many of studies had showed that the construction of various QS system depends on the type of bacteria. QS system exists widely in pathogenic bacteria, which build up the capability of infection, expression of toxic genes and pathogenesis. Therefore, it is a concerned topic in medicine realm that prevents and cures the diseases caused by pathogenic bacteria by targeting the QS system. Here, this review discussed the QS and its application in preventing and therapeutic effect for pathogenic bacteria.%细菌分泌一种或多种化学信号分子，这些化学信号分子作为诱导因子感知和判断菌群密度和周围环境的变化。

布鲁菌群体感应系统研究进展

摘要：布鲁茵病（布病）是世界上最严重的人畜共患病之一，布鲁菌作为布病的病原可以导致患病动物的流产及人类的马耳他热。群体感应系统对布鲁茵在胞内生存起到重要的作用，它调控着细菌的 Ⅳ 型分泌系统和鞭毛相关基因的表达。除此之外，布鲁菌群体感应系统在布鲁菌病的诊断与防治方面均有重要的价
革兰阳性菌中；第三类是以呋喃酰硼酸二酯为信号
菌中。
存在于革兰阴性菌和革兰阳性性菌和革兰阴性菌都通过群体感应系统与周围环境素的群体感应系统，
１布鲁菌的群体感应系统
布鲁菌是一种革兰阴性、兼性胞内寄生的短小
值。论文对布鲁菌群体感应系统的研究现状进行了系统的介绍。
关键词：布鲁菌；群体感应系统；细菌毒力
中图分类号：￥８５２．６１４文献标识码：Ａ文章编号：１００７ — ５０３８（２０１３）０２００８８０４
群体感应（ｑｕｏｒｕｍｓｅｎｓｉｎｇ，ＱＳ）系统是一种根
以寡肽类物质为信号素的群体感应系统，存在于在
据细菌密度调控基因表达的信号传递系统。革兰阳
进行信息交流。第１个群体感应系统是在费氏弧菌中发现的。ＮｅａｌｓｏｎＫＨ等口于１９７０年第一次报道了海洋费氏弧菌的菌体密度与生物发光呈正相关，而这种发光现象正是受到细菌自身的群体感应系统调控的。群体感应系统参与许多生物学过程，如生物发光，抗生素合成，细菌胞外酶和毒素的产生，生物膜的形成等。根据信号素的不同，细菌群体感应系统分为３种类型，一类是以酰基高丝氨酸内

群体感应-LASR

Revisiting the quorum-sensing hierarchy inPseudomonas aeruginosa:the transcriptionalregulator RhlR regulates LasR-specific factorsVale´rie Dekimpe and Eric De´zielCorrespondenceEric De´zieleric.deziel@iaf.inrs.caINRS-Institut Armand-Frappier,Laval,Que´bec H7V1B7,CanadaReceived29July2008 Revised11November2008 Accepted13November2008Pseudomonas aeruginosa uses the two major quorum-sensing(QS)regulatory systems las and rhl to modulate the expression of many of its virulence factors.The las system is considered to stand at the top of the QS hierarchy.However,some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions.Interestingly,such mutants arise spontaneously under various conditions,including in the airways of cystic fibrosis ing transcriptional lacZ reporters,LC/MS quantification and phenotypic assays,we have investigated the regulation of QS-controlled factors by the las system.Our results show that activity of the rhl system is only delayed in a lasR mutant,thus allowing the expression of multiple virulence determinants such as pyocyanin,rhamnolipids and C4-homoserine lactone(HSL)during the late stationary phase.Moreover,at this stage,RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions,including production of3-oxo-C12-HSL and Pseudomonas quinolone signal(PQS).P.aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over.This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.INTRODUCTIONPseudomonas aeruginosa is a ubiquitous and versatile bacterium involved in numerous pathogenic infections affecting immunocompromised individuals and those suffering from cystic fibrosis(Marshall&Carroll,1991; Pier,1985;Speert,1985).This bacterium regulates most of its virulence determinants in a cell-density-dependent manner via a mechanism called quorum-sensing(QS). Such global regulatory systems are found in most bacterial species,and control several and diverse biological func-tions,such as virulence,bacterial conjugation,biolumin-escence and biofilm formation(de Kievit&Iglewski,2000; Donabedian,2003;Loh et al.,2002;Miller&Bassler,2001). QS is mediated by diffusible signalling molecules released into the external environment.These signals,when reach-ing specific concentrations correlated with specific popu-lation cell densities,bind to and activate their respective transcriptional regulators.In P.aeruginosa,two conven-tional complete QS systems are known:the synthases LasI and RhlI produce the N-acylhomoserine lactones3-oxo-C12-HSL and C4-HSL respectively,which induce their cognate LuxR-type transcriptional regulators LasR and RhlR,responsible for the activation of numerous QS-controlled genes(Juhas et al.,2005;Pesci et al.,1997). Among genes activated by these two regulators are those coding for the LasI and RhlI synthases.Since N-acyl-HSLs induce their own production,they are called autoinducers. More recently,a third,distinct QS system has been unveiled.It is composed of a transcriptional regulator from the LysR family,MvfR(PqsR),which directly activates two operons(phnAB and pqsABCDE)required for the biosynthesis of4-hydroxy-2-alkylquinolines (HAQs),including molecules involved in4-quinolone signalling(De´ziel et al.,2004;Le´pine et al.,2004;Pesci et al., 1999),and for the activation of many QS-controlled genes, via pqsE(De´ziel et al.,2005;Diggle et al.,2006;Farrow et al.,2008).Among the HAQs,4-hydroxy-2-heptylquino-line and the Pseudomonas quinolone signal(PQS)act as activators of the MvfR regulator,inducing a positive feedback loop typical of QS systems(Xiao et al.,2006a). QS regulation is a very complex and extensive network influencing,both positively and negatively,the transcrip-tion of perhaps5–10%of the P.aeruginosa genome (Hentzer et al.,2003;Schuster et al.,2003;Wagner et al., 2003).The LasR regulator is known to initiate the QS regulatory system,as it activates the transcription of a number of other regulators,such as rhlR,defining aAbbreviations:HAQ,4-hydroxy-2-alkylquinoline;HSL,homoserinelactone;PQS,Pseudomonas quinolone signal;QS,quorum sensing.Two supplementary figures are available with the online version of thispaper.Microbiology(2009),155,712–723DOI10.1099/mic.0.022764-0 712022764G2009SGM Printed in Great Britainhierarchical QS cascade from the las to the rhl regulons (Latifi et al.,1996;Pesci et al.,1997).Over the last few years, many whole-genome transcriptomic studies have been published with the aim of identifying genes that are under the control of LasR and/or RhlR(Hentzer et al.,2003; Rasmussen et al.,2005;Schuster et al.,2003;Wagner et al., 2003).Specific directly activated genes were clearly identified as belonging to the rhl regulon,such as rhlAB(rhamnolipid biosynthesis),lecA(lectin),hcnABC(HCN production)and both phzABCDEFG operons(phenazine biosynthesis)(Latifi et al.,1995;Schuster et al.,2004;Schuster&Greenberg, 2007;Whiteley et al.,1999;Winzer et al.,2000).However, the situation is not as clear for many LasR-controlled genes, for which it has not been possible to define a single consensus LasR binding site sequence in the promoter region,suggesting that some of these genes are activated indirectly(Schuster et al.,2004;Schuster&Greenberg, 2007).Actually,most QS-regulated factors are more or less influenced by both LasR and RhlR,as is the case for the proteases LasA(staphylolytic protease)and LasB(elastase) (Freck-O’Donnell&Darzins,1993;Hentzer et al.,2003; Nouwens et al.,2003;Schuster et al.,2003;Toder et al.,1994; Wagner et al.,2004).Thus QS plays a predominant role in the regulation of virulence determinants in P.aeruginosa. Surprisingly,however,there are increasing reports that lasR mutants occur frequently in the natural environment (Cabrol et al.,2003),in airways from individuals with cystic fibrosis(D’Argenio et al.,2007;Smith et al.,2006),in intubated patients(Denervaud et al.,2004)and in individuals suffering from bacteraemia,pneumonia or wound infection(Hamood et al.,1996).This is intriguing, since the LasR regulator is widely considered essential for full P.aeruginosa virulence(Preston et al.,1997;Rumbaugh et al.,1999;Storey et al.,1998).The LasR transcriptional regulator is generally considered to sit at the top of the QS hierarchy in P.aeruginosa(Latifi et al.,1996).However,we and others have observed that the phenazine pyocyanin is overproduced by lasR mutants at the late stationary phase(De´ziel et al.,2005;Diggle et al., 2003).As shown in Fig.1,a lasR mutant produces less pyocyanin during early growth phases,although at the end of exponential growth and during early stationary phase, pyocyanin begins to be produced.During late stationary phase,after24h of cultivation,the lasR mutant cultures contain much more pyocyanin than cultures of the wild-type strain(35mg l21compared to2.5mg l21,respect-ively).This is unexplained,since pyocyanin production is known to be regulated by QS(Latifi et al.,1995).The regulator of the pyocyanin biosynthesis genes(phz genes)is RhlR(Brint&Ohman,1995),whose transcription is considered to require LasR(de Kievit et al.,2002;Latifi et al.,1996;Pearson et al.,1997;Pesci et al.,1997).In theory,pyocyanin production is thus expected to be absent in lasR mutants,whereas experimental data show that it is actually only delayed(Fig.1).In order to better understand the specific role of LasR and its involvement in expression of virulence factors,we have characterized the expression of QS-controlled determinants in a lasR mutant and have observed that during stationary phase,many QS-regulated virulence factors are expressed. Our data show that at this stage of growth,the RhlR regulon is activated.Moreover,we found that RhlR is able to induce LasR-regulated genes(including some consid-ered specific such as lasI)in the absence of lasR,unveiling a new mechanism for the bacteria to bypass a defect in their QS regulation,allowing RhlR to induce the las system when LasR is non-functional.METHODSStrains,plasmids and growth conditions.Table1lists strains and plasmids.Bacteria were routinely grown in Tryptic Soy Broth(TSB) medium at37u C in a roller drum,with appropriate antibiotics when required(carbenicillin300mg l21and tetracycline75mg l21for P. aeruginosa;carbenicillin100mg l21and tetracycline15mg l21for Escherichia coli).TSB plates contained1.5%agar.For pyocyanin and rhamnolipid detection,King’s A medium was used(King et al.,1954). All measurements of optical density and absorbance were obtained with a Thermo Scientific NanoDrop1000spectrophotometer.An isogenic lasR rhlR double mutant was generated by allelic exchange of the rhlR gene in a lasR background with pSB224.10A using sucrose counterselection(Beatson et al.,2002).2040608010201101020Pyocyaninconcn(mgl_1)Time (h)Time (h)Growth(OD6)Fig.1.Expression of pyocyanin is delayed in alasR mutant:P.aeruginosa lasR mutant con-taining a constitutive rhlR(pUCPSK rhlR)orlasR(pUCPSK lasR)expression vector,or thesame vector without rhlR or lasR(pUCPSK),compared with the wild-type and the lasR rhlRmutant.Revisiting quorum sensing in P.aeruginosa 713Standard methods were used to manipulate DNA.Plasmid pDN19 (Nunn et al.,1990)was used to construct pVD1,containing the lasI gene under its own promoter.A region spanning from305bp upstream to170bp downstream of the lasI ORF was amplified and inserted between the Xba I and Hin dIII sites in the pDN19multiple cloning site.The gene fragment was generated from genomic DNA using PCR with primers59-GCTCTAGATTTTGGGGCTGTGTTC-TCTC-39and59-CCCAAGCTTACTCGAAGTACTGCGGGAAA-39. The construction was confirmed by effective complementation of a lasI mutant.Plasmids were introduced by electroporation(Choi et al., 2006).lasR mutant subcultures were carried out as follows:a first preculture was made at day1and used to inoculate fresh medium for day2;the latter was used to inoculate fresh medium for day3.Pyocyanin was measured during each day of culture.b-Galactosidase activity assay.Bacteria containing the gene reporter fusions were routinely grown overnight from frozen stocks in TSB with appropriate antibiotics,then subcultured in triplicate at a starting OD600of0.05without antibiotic.Culture samples were regularly taken for determination of growth(OD600)and b-galactosidase activity(Miller,1972).N-Butyryl-L-homoserine lactone (C4-HSL)was purchased from Sigma-Aldrich and the stock solution prepared in acetonitrile.Quantification of rhamnolipids,pyocyanin,AHLs and HAQs. Detection and measurements were performed by LC/MS.For pyocyanin,AHLs and HAQs,480m l culture samples were taken at regular intervals,used for determination of growth(OD600),and mixed with120m l acetonitrile containing50mg l215,6,7,8-tetradeutero-PQS for a final concentration of10mg l21as internal standard.After centrifugation,20m l aliquots of the supernatants were directly injected for LC separation on an Agilent HP1100HPLC system equipped with a36150mm C8Luna reverse-phase column (Phenomenex).A1%acidified water/acetonitrile gradient was used as the mobile phase at a flow rate of0.4ml min21,split to10%with a Valco Tee.A Quattro II(Waters)triple-quadrupole MS was used for molecule detection.Data acquisition was performed in full scan mode with a scanning range of130–350Da.Precise quantification of C4-HSL and3-oxo-C12-HSL was performed by MS/MS,as described previously(De´ziel et al.,2005).For rhamnolipid quantification, 500m l culture samples were taken at regular intervals,used for determination of growth(OD600),and diluted with an equivalent volume of methanol.After centrifugation,20m l aliquots of the supernatants were injected for LC/MS analysis as described pre-viously,using16-hydroxyhexadecanoic acid as internal standard (De´ziel et al.,1999;Le´pine et al.,2002).Elastase and protease enzymic assays.TSB plates supplemented with1%skim milk were inoculated with10m l from cultures at OD6003. Plates were incubated at37u C for3days.For specific LasB elastolytic activity,we used a protocol adapted from that of Bjorn et al.(1979). Briefly,filter-sterilized culture supernatant samples(100m l)from late stationary phase cultures were mixed with5mg elastin Congo red (Sigma)and300m l0.1M Tris/HCl pH7.2.Release of Congo red from degraded elastin was measured as A495after2h of incubation at37u C followed by centrifugation.For assessment of LasA staphylolytic activity, 4.5ml of Staphylococcus aureus overnight cultures were boiled for 15min.and100m l was mixed with300m l of filtered culture supernatants.The OD600was measured after2h of incubation at 37u C with agitation.All experiments were carried out in triplicate. RESULTSThe expression of RhlR-regulated factors is only delayed in the absence of LasRBased on previous observations reporting late pyocyanin production in lasR mutants,we decided to investigate theTable1.Bacterial strains and plasmids used in this studyStrain or plasmid Characteristics Source or reference BacteriaE.coli DH5a supE44D lacU169(w80lacZ D M15)hsdR17recA1endA1gyrA96thi-1relA1Hanahan(1983)P.aeruginosa/lab no.:PA14/ED14Clinical isolate UCBPP-PA14Rahme et al.(1995)PA14lasR/ED69lasR::Gm derivative of ED14De´ziel et al.(2004)PA14lasR rhlR/ED266rhlR::Tc derivative of ED69This studyS.aureus Newman Laboratory strain ATCC25904PlasmidspMIC61(pUCPSK-lasR)lasR in pUCPSK with lac promoter as a Hin dIII–Eco RI fragment(59–39lasR)John Mattick,Institute of Molecular Bioscience,University of Queensland, AustraliapMIC62(pUCPSK-rhlR)rhlR in pUCPSK with lac promoter as a Hin dIII–Eco RI fragment(59–39rhlR)John MattickpPCS1002pLP170containing rhlR-lacZ Pesci et al.(1997) pSB224.10A pRIC380suicide vector carrying rhlR::Tc Beatson et al.(2002) pVD1pDN19containing lasI with its native promoter,Tc r This studypME3853pME6010with a174bp lasI upstream fragment and translationallasI::lacZ fusion containing the first13lasI codons,Tc rPessi et al.(2001) pUCPSK E.coli–P.aeruginosa shuttle vector Watson et al.(1996)pLJR50lasB p-lacZ transcriptional reporter fusion;contains nt2190to+4of the lasB promoter region,Cb r Toder et al.(1994)V.Dekimpe and E.De´ziel714Microbiology155mechanism involved in this phenomenon,as an introduc-tion to exploring QS during the stationary phase.Since RhlR is the known regulator of the phz genes,we hypothesized that late pyocyanin production is due to RhlR activity.In the absence of lasR,RhlR should activate the expression of the phz genes in the late stationary phase,and in its absence,no pyocyanin should be produced.As shown in Fig.1,unlike the lasR mutant,the lasR rhlR double mutant does not produce this phenazine at all.Moreover, lasR(pUCPSK-rhlR),which constitutively expresses rhlR from a plasmid,produces pyocyanin at the same time as the wild-type,confirming that RhlR is responsible for the timing of pyocyanin production.As expected,continued expression of rhlR results in higher production of sR(pUCPSK)acts like the lasR mutant, confirming that the vector does not influence pyocyanin expression.Finally,lasR(pUCPSK lasR)does not over-produce pyocyanin,unlike the lasR mutant,showing that the lasR mutation is responsible for this phenotype.It is also noteworthy that a lasI mutant shows the same pyocyanin overproduction phenotype as the lasR mutant (data not shown).To ensure that optical density during all growth stages,and particularly during stationary phase, truly reflected the number of living bacterial cells,we also determined the viable cell counts.This showed that the growth rates and survival of the lasR mutant and the wild-type were essentially the same(see Supplementary Fig.S1a, available with the online version of this paper),thus confirming that the difference in pyocyanin production is not the result of variations in the number of viable cells. To ensure that this late pyocyanin production was not due to a spontaneous mutation that might have occurred in the lasR background,we subcultured a culture of the lasR mutant on three consecutive days in fresh medium,every time monitoring the production of pyocyanin. Consistently,the cultures had to reach the late stationary phase before producing pyocyanin,indicating that this phenotype in not due to accumulation of secondary mutations during cultivation(see Supplementary Fig.S1b). If RhlR is present and active during the late stationary phase in a lasR mutant,then we should be able to detect RhlR-regulated factors other than pyocyanin.The rhlAB and rhlC genes,coding for enzymes involved in rhamno-lipid biosynthesis,and rhlI,coding for the C4-HSL synthase,are all directly regulated by RhlR(de Kievit et al.,2002;Medina et al.,2003).We precisely quantified rhamnolipids and C4-HSL in lasR,lasR rhlR and lasR(pUCPSK-rhlR)cultures.As shown in Fig.2(a,b), the lasR rhlR double mutant was unable to synthesize rhamnolipids or C4-HSL,while the lasR mutant produced these molecules with a delay,essentially in late stationary phase.These results support the hypothesis thatexpression 1020304010201101020102011010200.40.30.20.1Rhamnolipidconcn(mgl_1)C4-HSLconcn(mgl_1)Growth(OD6)Growth(OD6)(a)(b)Time (h)Time (h)Time (h)Time (h)Fig.2.Expression of RhlR-controlled factorsis delayed in a lasR mutant.P.aeruginosawild-type and lasR mutant containing ornot a constitutive rhlR expression plasmid(pUCPSK-rhlR)are compared.Production of(a)rhamnolipids and(b)C4-HSL.Revisiting quorum sensing in P.aeruginosa 715of the rhl regulon is only delayed in a lasR mutant.The production of C4-HSL and rhamnolipids was restored to levels similar to wild-type when the lasR mutant was transformed with an rhlR expression vector,confirming that RhlR is responsible for these phenotypes.These results show that the delayed expression of RhlR-controlled phenotypes in a lasR background can be restored by expressing rhlR.In order to obtain additional evidence that RhlR is indeed expressed in a lasR mutant,we evaluated the transcription of rhlR with a lacZ fusion reporter.As shown in Fig.3, maximal rhlR transcription occurs at the early stationary phase in the wild-type strain.Furthermore,it follows a similar expression pattern in the lasR mutant background, but at lower levels.Still,during late stationary phase,level of rhlR expression slightly increases in the lasR mutant, while it decreases in the wild-type.These data support the significant presence of RhlR in lasR mutants during late stationary phase,as previously reported(Diggle et al., 2003).It is well established that the production of proteolytic enzymes such as LasA and LasB,responsible for staphylo-lytic and elastolytic activities respectively,is under LasR regulation(Rust et al.,1996;Storey et al.,1998;Toder et al., 1991).However,there are indications that production of these enzymes might also be under partial RhlR control (Brint&Ohman,1995;Diggle et al.,2003;Pearson et al., 1997).To evaluate global protease activity of the strains, the wild-type strain,and lasR and lasR rhlR mutants,were inoculated on solid medium containing skim milk. Protease activity was visible for the lasR mutants while the double mutant was unable to degrade milk proteins (see Supplementary Fig.S2).Since this test only indicates general proteolytic activity,it was interesting to target specific proteases.Fig.4(a)shows that the lasR mutant is able to activate lasB expression late in stationary phase, while the double lasR rhlR mutant cannot.Detection of LasB activity confirmed these results.During late stationary phase,the lasR mutant shows significant elastolytic activity, which is nearly as high as that in lasR(pUCPSK-rhlR) (Fig.4b).Finally,Fig.4(c)shows that the wild-type and the lasR mutant,complemented with rhlR or not,express LasA activity,while the lasR rhlR double mutant does not.Taken together,all these results indicate that the expression of many QS-controlled factors is only delayed when LasR is defective.RhlR controls factors generally considered to be solely regulated by LasRAnother observation we and others have made is that not only pyocyanin but also PQS is produced during late stationary phase by a lasR mutant(De´ziel et al.,2004; Diggle et al.,2003).This was unexpected,since the final step in PQS synthesis is catalysed by the lasR-dependent PqsH enzyme(De´ziel et al.,2004;Gallagher et al.,2002; Whiteley et al.,1999;Xiao et al.,2006b).It is of note that there is a close correlation between the timing of production of both PQS and pyocyanin in lasR mutant backgrounds(De´ziel et al.,2005;Diggle et al.,2002,2003). To test if RhlR might also be responsible for this effect,we quantified PQS production by the wild-type and the lasR, lasR rhlR and lasR(pUCPSK-rhlR)mutants.As shown in Fig.5(a),during the exponential and early stationary growth phases,PQS production is totally absent in the double mutant and barely detectable in the lasR mutant unless rhlR is expressed,which leads to a substantial reduction in the delay observed for that mutant.The same reduction of PQS is observed in a lasI mutant,and can also be restored by overexpressing RhlR in that mutant(data not shown).At the late stationary phase,however,the concentration of PQS in lasR mutant cultures is similar to the wild-type,while the double mutant still shows no detectable production.These data explain the late PQS production in a lasR mutant by the activity of RhlR.We then asked whether lasI,probably the most specific LasR-regulated gene,which codes for the autoinducer synthase producing3-oxo-C12-HSL,might also be regulated by RhlR.As expected from the above data,we found that3-oxo-C12-HSL production is greatly increased in lasR(pUCPSK-rhlR)compared to the wild-type strain,at the same optical density(Fig.5b).It also shows that3-oxo-C12-HSL is eventually produced in a lasR mutant at late stationary phase,but is totally absent if rhlR is alsodefective.16111621261020110Time (h)1020Time (h)13×b-Galactosidaseactivity(Millerunits)Growth(OD6)Fig. 3.rhlR transcription in a lasR mutantincreases during late stationary phase.b-Galactosidase activity using the pSC1002vector containing the rhlR-lacZ transcriptionalreporter.V.Dekimpe and E.De´ziel716Microbiology15514001200100080060040020010201020G r o w t h (O D 600)101lasB -lacZ654321E l a s t o l y t i c a c t i v i t y (A 495)S . a u r e u s l y s i s (%)80604020Time (h)Time (h)LasR PA14lasR rhlRWild-typelasRlasR rhlRlasR (pUCPSK-rhlR )Wild-type lasR lasR rhlRlasR (pUCPSK-rhlR )(a)(b)(c)b -G a l a c t o s i d a s e a c t i v i t y (M i l l e r u n i t s )sA and LasB are activated late in a lasR mutant but not in a lasR rhlR double mutant.(a)Transcription of the lasB gene;(b)elastolytic (LasB)activity;(c)staphylolytic (LasA)activity.50.40.30.20.11015202530102010201020110P Q S p r o d u c t i o n (m g l _1)3-O x o -C 12-H S L p r o d u c t i o n (m g l _1)G r o w t h (O D 600)G r o w t h (O D 600)Time (h)Time (h)Time (h)Time (h)(a)(b)Fig.5.Production of PQS (a)and 3-oxo-C 12-HSL (b)requires rhlR in the absence of lasR .LC/MS analysis from culture supernatants.Revisiting quorum sensing in P.aeruginosa717RhlR controls lasI in a heterologous system In order to further identify RhlR as an alternative activatorof lasI transcription in the absence of a functional LasR,we constructed a heterologous system in E.coli .A vector (pME3853)carrying the lasI-lacZ gene reporter was introduced into E.coli DH5a .In the presence of the rhlR gene constitutively expressed on another compatible plasmid,and with addition of its autoinducer C 4-HSL,b -galactosidase activity was greatly enhanced in the E.coli strain,while only basal expression was detected in absence of rhlR or C 4-HSL (Fig.6a).To confirm 3-oxo-C 12-HSL production through activation by RhlR,a vector contain-ing lasI under its native promoter was introduced into E.coli DH5a .3-Oxo-C 12-HSL was detected in this heterologous system only in the presence of both RhlR and its autoinducer C 4-HSL (Fig.6b).DISCUSSIONP.aeruginosa is an opportunistic pathogen that relies on its impressive ability to coordinate gene expression in order to compete against other species for nutrients or colonization.QS appears essential for this bacterium for competitiveness in clinical or environmental niches.The QS LasR transcriptional regulator is known to control a wide array of P.aeruginosa virulence-associated factors.Nevertheless,several reports mention the high frequency of lasR mutations among clinical and environmental isolates (Cabrol et al.,2003;D’Argenio et al.,2007).Most intriguingly,some lasR mutants still produce QS-regulated virulence factors such as pyocyanin (Heurlier et al.,2005),and naturally occurring lasR mutants have been isolated from wounds or intubated patients (Denervaud et al.,2004;Hamood et al.,1996).It was thus interesting to analyse the involvement of LasR in the expression of QS-regulated virulence determinants in more detail.This study provides new insights into the interplay between the las and the rhl QS systems in P.aeruginosa ,and demonstrates that a lasR mutation does not lead to loss of virulence factors.Expression of the rhl regulon is delayed until the late stationary phase in a lasR mutant,and is thus responsible for the late production of virulence factors in this background,such as pyocyanin,QS signalling molecules and proteases.These observations provide a solid basis allowing us to explain numerous inconsistencies in previous reports,and bring some clarifications to the P.aeruginosa QS model,as summarized in Fig.7.RhlR-regulated factors are expressed late in a lasR mutantThe delayed production of pyocyanin in a lasR mutant background has been anecdotally observed in numerousreports (De´ziel et al.,2005;Diggle et al.,2002,2003;Heurlier et al.,2005;Kohler et al.,2001;Lujan et al.,2007;Salunkhe et al.,2005).It has been suggested that RhlR might be involved in that production,although no evidence was presented (Diggle et al.,2003).Here we present evidence for the role of the RhlR regulator in pyocyanin production in a lasR mutant,since no production can be observed in a lasR rhlR double mutant and production is advanced in a lasR mutant comple-mented with rhlR.The activity of RhlR during stationary phase in a lasR mutant was confirmed by the delayed production of other RhlR-controlled factors,C 4-HSL and rhamnolipids.Others3530252015105b -G a l ac t o s id a se a c t i v i t y (M i l l e r u n i t s )3-O x o -C 12-H S L c o n c n (m g l _1)0.120.100.080.060.040.02DH 5a(p V D1)DH5a (pME 3853)DH5a(pM E 3853)+C4-H S LDH5a (pME 3853)(pU C P S K -r h l R )DH5a (p M E 3853)(p U C P S K -r h l R )+C 4-H S L D H 5a (p V D 1)(p U C P S K -r h l R )D H 5a (p V D 1)(p U C P S K -r h l R )+C 4-H S L(a)(b)sI is activated by RhlR in a heterologous E.coli DH5a system in the presence of either or both C 4-HSL (5mg l ”1)and rhlR (pUCPSK-rhlR ).(a)lasI-lacZ expression (pME3853);(b)3-oxo-C 12-HSL production in the presence of the lasI gene with its native promoter (pVD1).V.Dekimpe and E.De´ziel 718Microbiology 155。

山东大学科技文献写作论文群体感应及其在合成生物学中的应用

群体感应及其在合成生物学中的应用胡斯琪 2011级生科2班 201100140114摘要：细菌的群体感应现象由于其独特的作用机制正越来越引起人们的关注，这种细胞与细胞之间通过种群密度而感知信息的交流方式使得细菌可以在群体的规模上应对外界的变化。

群体感应由于能够实现细胞行为的同步化，故在很多领域都有广泛的应用，特别是在新兴的合成生物学领域。

本文主要阐述了群体感应的几种方式及其作用机理，并简要的介绍了群体感应在合成生物学上的应用，最后对群体感应系统做了进一步的展望。

1引言：细胞与细胞之间的通讯通常被认为是真核细胞的专利，但是近年来的研究发现，细菌与细菌之间能通过一种被称作自体诱导物的小分子的类激素有机化合物来进行交流，这种行为被称为群体感应(Quorum sensing)[1]。

细菌的群体感应现象最早是来自于有关费氏弧菌（Vibrio fischeri）的生物发光（bioluminescence）机制的报道[2]，费氏弧菌能合成一种酰基高丝氨酸内脂类（acyl-homoserine lactone, AHL）的信号分子，可以自由扩散至细胞外，随细胞浓度的增加而增加。

当其达到一定浓度时，该AI分子与胞内的LuxR分子结合，促使其识别发光酶基因的启动子，从而启动与发光相关的基因的表达。

后来研究发现这种群体感应系统不仅在费氏弧菌中存在，还在许多革兰氏阳性菌和革兰氏阴性菌甚至是真核生物中出现，至今，群体感可分为种内QS系统和种间QS系统，种内QS系统又分革兰氏阳性菌和革兰氏阴性菌的系统。

细菌的群体感应被描述为细菌通过密度调节基因的表达的机制，它可以帮助细胞计算邻近种群的细胞数量以在群体的规模上对外界刺激产生应答作用[3]。

群体感应是细胞间通讯的重要方式，它是指单个细胞通过自身合成的自体诱导物(Autoinducer)的富集来感知菌群密度的现象。

当自诱导剂的浓度随着细菌密度的增加达到特定阈值时，某些基因像开关一样被打开，启动后续一系列基因的表达[4]。

细菌共培养及其系统中群体感应现象的研究进展

２．大连民族学院生命科学学院，辽宁大连１１６６００；３．西南大学食品学院，重庆４００７１５）
摘要：本文对微生物的共培养方式、分类、发展及应用进行了简介，并对不同种属的微生物共培养后产生的作用进行
了综述。微生物共培养系统不仅受到协同代谢作用的调控，群体感应在微生物共培养中也扮演着重要的角色。与纯培养相比，共培养体系生物被膜的形成、信号分子和毒力因子的产生、细菌素的合成以及胞外蛋白酶的分泌等都有所不同。研究群体感应的作用机理有助于共培养技术的进一步发展与应用。
ｅｆｆｅｃｔｓｏｆｃｏ — ｃｕｌｔｕｒｅｏｆｄｉｆｆｅｒｅｎｔｓｐｅｃｉｅｓｗｅｒｅｓｕｍｍａｒｉｚｅｄ．Ｍｉｃｒｏｂｉａｌｃｏ —ｃｕｌｔｕｒｅｓｙｓｔｅｍｗａｓｒｅｇｕｌａｔｅｄｎｏｔｏｎｌｙｂｙｔｈｅｃｏｌｌａｂｏｒａｔｉｖｅｍｅｔａｂｏｌｉｓｍ，ｂｕｔａｌｓｏｂｙｑｕｏｒｕｍｓｅｎｓｉｎｇｉｎｍｉｃｒｏｂｉａｌｃｕｌｔｕｒｅｓ．Ｃｏｍｐａｒｅｄｗｉｔｈｔｈｅｐｕｒｅｃｕｌｔｕｒｅ，ｂｉｏｆｉｌｍｆｏｒｍａｔｉｏｎ，ｓｉｇｎａｌｉｎｇｍｏｌｅｃｕｌｅｓ，ｖｉｒｕｌｅｎｃｅｆａｃｔｏｒｓｏｆｐｒｏｄｕｃｔｉｏｎ，ｂａｃｔｅｒｉｏｃｉｎｓｙｎｔｈｅｓｉｓａｎｄｓｅｃｒｅｔｉｏｎｏｆｅｘｔｒａｃｅｌｌｕｌａｒｐｒｏｔｅａｓｅｏｆｃｏ —ｃｕｌｔｕｒｅｓｙｓｔｅｍｗｅｒｅｄｉｆｆｅｒｅｎｔ．Ｔｈｅｒｅｓｅａｒｃｈｏｆａｃｔｉｏｎｍｅｃｈａｎｉｓｍｏｆｑｕｏｒｕｍｓｅｎｓｉｎｇｗｏｕｌｄｉｍｐｒｏｖｅｆｕｒｔｈｅｒｄｅｖｅｌｏｐｍｅｎｔａｎｄａｐｐｌｉｃａｔｉｏｎｏｆｃｕｌｔｉｖａｔｉｏｎｔｅｃｈｎｏｌｏｇｙ．Ｋｅｙｗｏｒｄｓ：ｍｉｃｒｏｏｒｇａｎｉｓｍｓ；ｃｏ－ｃｕｌｔｕｒｅ；ｑｕｏｒｕｍｓｅｎｓｉｎｇ；ｓｙｎｅｒｇｙｍｅｔａｂｏｌｉｓｍ

革兰氏阴性菌群体感应抑制剂的研究进展

作用的过程中并不影响细菌的生长，因此不会引
天然来源主要包括植物，动物和微生物。其中植物来源和微生物来源的ＱＳＩｓ占到天然来源
ＱＳＩｓ的绝大部分。１．１．１植物来源ＱＳＩｓ
目（ＬＹ１６Ｈ３ｏｏｏ０８）。
作者简介：王鸿（１９７２一），女，黑龙江齐齐哈尔人，教授，博士生导师，研究方向为药学。Ｅ－ｍａｉｌ：ｈｏｎｇｗ＠ｚｊｕｔ．ｅｄｕ．ｃａ。
一
然来源和化学合成来源。天然来源ＱＳＩｓ通常具有
修回日期：２０１６ — ０４ — ０８基金项目：国家自然科学基金资助项目（２１３３７００５），国家自然科学基金资助项目（３０９７３６８１），浙江省自然科学基金资助项
植物来源的ＱＳＩｓ研究较为广泛，最早发现海
起细菌耐药性的产生。这一新颖的抑菌机制使其
在开发新型抗感染药物方面有很大潜力。
洋红藻（Ｄｅｌｉｓａｐｕｌｃｈｒａ）中的溴代呋喃酮（５ｚ）一４一
ｒａｎｏｎｅ（１）可以阻断哈氏弧菌的生物荧光的产生Ｉ２Ｊ。木耳ｕｒｉｃｕｌａｒｉａａｕｒｉｃｕｌａｒ）色素提取物，枇杷树中的鞣质成分，小茴香（Ｃｕｍｉｎｕｍｃｙｍｉｎｕｍ）中的甲基丁香酚１，２－ｄｉｍｅｔｈｏｘｙ一４一（２一ｐｒｏｐｅｎ一１一ｖ１）

细菌群体感应在微生物生态系统中的作用研究

细菌群体感应在微生物生态系统中的作用研究细菌群体感应是一种自协调的细菌行为，细菌通过分泌信号分子来与它们周围的同种细菌进行通信，并协同地做出响应。

这种协作行为有助于建立细菌社区，并有助于它们在复杂的微生物生态系统中生存和繁殖。

本文将讨论细菌群体感应在微生物生态系统中的作用，并探讨该领域目前的研究进展。

1. 细菌群体感应的基本原理细菌群体感应是一种通过细菌间分泌的信号分子进行交流的行为，这些分子可以传递不同的信息，例如细胞密度、群体方向、环境变化等。

在感应过程中，当一定数量的信号分子被积累到足够数量时，细菌将协调做出共同的行为。

例如，一些细菌会通过群体感应来形成生物膜，从而形成细菌社区，或者来协同合成一些生物活性物质，如光合色素、激素、抗生素等。

这些共同的行为有助于细菌在微生物生态系统中生存和繁殖。

2. 细菌群体感应在微生物生态系统中的作用细菌群体感应在微生物生态系统中起着至关重要的作用。

首先，它有助于细菌建立稳定的细菌社区，并与其他细菌、微生物甚至宿主紧密相连。

这些细菌社区有时会形成生物膜，从而能够更好地抵御环境压力。

其次，它有助于细菌在微生物生态系统中发挥“分工协作”的作用，不同种类的细菌能够通过群体感应来分布不同的环境和角色，以最大化资源利用率并优化生态系统。

另外，细菌群体感应还发挥着各种生态学角色。

例如，在土壤微生物系统中，细菌群体感应可以促进植物生长和根际土壤释放养分。

一些细菌群体感应所产生的代谢产物还被发现对宿主免疫反应和免疫功能具有重要意义。

此外，细菌群体感应还被认为是生态系统中细菌和其他生物之间相互作用的重要媒介，它能够帮助生物维持相互联系并参与生态系统的稳定性。

3. 细菌群体感应的研究进展目前，细菌群体感应的研究进展日新月异。

这是因为细菌群体感应在医学、环境保护、农业等领域都有重要应用价值。

例如，在医学中，对细菌群体感应的深入研究能够有助于探索新型抗生素的生产和应用；环境保护中，它可以帮助减少有毒物质的生产和释放，改善微生物生态环境；在农业中，它能够协助控制农业害虫和植物病害。

细菌群体感应的研究进展

信息交流是通过小的、可扩散的信号分子（自我诱导物）来完成。文章阐述自我诱导物的结构与合成，
并讨论几种革兰氏阴性细菌的群体感应模式、生理特性及其调控机制。关键词：细菌；群体感应；自我诱导物；生理特性
维普资讯
Ｖ６卷增刊第２
ｏ．６，Ｓｐ１２ｕ．
广西业生物学农ｉ．ｎｉｉｎｃｅ ● ［ＳＪｕｒｌｏｕａｎｇｘｉＡｇｒｃａｄＢｏ● ｃｅｏｎａｆＧ
２００７年６月
Ｊｎ．０７ｕｅ２０
ｃｍｍｕｎｃｔｏ［－ｏｉａｉｎ）１］３
。
（ｅｌｅｌＣｌｃｌ —
收稿日期２００７—００；４— ９作者简介韦珂
修回日期：２００７—０５—２。２
基金项目广西大学科研基金项目（１０５）ＤＤ０８。６（７）１２一，女，广西宜州人．讲师，博士；Ｅ— ｉ：ｗｅｋｆｏｒｙｈｏｃｍ．ｎ。９ｍａｌｉｅｌｗｅ＠ａｏ．ｏｃ
维普资讯
广
西
农
业
生
物
科
学
第２６卷
当单个细菌向周围环境中释放自我诱导物时，自我诱导物的浓度非常低，甚至不能被检测出
ＡｂｔａｔＱｕｒｍｅｓｎｓｍｅｉｔｄｔｒｕｈａｐｏｕｔｏｆｄｆｕｉｌｉｎｌｍｏｅｕｅｓｒｃ：ｏｕｓｎｉｇｉｄａｅｈｏｇｒｄｃｉｎｏｉｓｂｅｓｇａｆｌｃｌｓ

革兰氏阴性菌群体感应系统研究进展

革兰氏阴性菌群体感应系统研究进展摘要：群体感应（quorum sensing，QS），又称为自体诱导(autoinduction)，是一种调节细菌群居行为及特殊基因表达的有效机制，描述细菌之间保持细胞密度变化的化学信号，是一种细菌与细菌间的通讯系统。

通常将群体感应系统分为革兰氏革兰氏阴性菌的LuxI/LuxR型QS系统、革兰氏阳性菌的寡肽类群体感应系统和感知种间信号的群体感应系统。

植物病原细菌中常见的致病菌是革兰氏阴性菌，所以对革兰氏阴性菌群体感应系统的研究很有必要。

关键词：群体感应；革兰氏阴性；LuxI/LuxR群体感应（quorum sensing，QS），又称为自体诱导(autoinduction)，是一种调节细菌群居行为及特殊基因表达的有效机制，描述细菌之间保持细胞密度变化的化学信号，是一种细菌与细菌间的通讯系统。

这种通讯系统依赖于一种小的可扩散的信号分子，这种小的信号分子称为自体诱导素(autonicers,AI)，由细菌产生并向细胞外扩散，在周围环境中积累。

随着种群密度的增加，环境中积累的AI信号分子的浓度也成比例地增高，当达到一定阈值水平时细菌通过细胞内受体对这些信号分子进行检测，进而子与一种转录激活因子结合，诱导有关基因的协调表达[1]。

自体诱导物与转录活性蛋白相互作用，启动基因表达，调节相关群落活动和独立过程。

目前已经在细菌中发现了129个与群体感应相关的基因，包括群体感应调节基因和信号合成基因。

通常将群体感应系统分为革兰氏革兰氏阴性菌的LuxI/LuxR型QS系统、革兰氏阳性菌的寡肽类群体感应系统和感知种间信号的群体感应系统。

植物病原细菌中常见的致病菌是革兰氏阴性菌，所以对革兰氏阴性菌群体感应系统的研究很有必要。

在革兰氏阴性细菌中存在自身常见的LuxI/LuxR型QS系统和感知种间信号的AI-2信号系统。

1 革兰氏阴性菌的LuxI/LuxR型QS系统革兰氏阴性菌中感知种内数量的QS系统一般利用酰基高丝氨酸内酯(N-acyl-homoserinelactones,简称acyl-HSL或AHLs，这类分子一般称为AI-1。

铜绿微囊藻群体感应的初步研究

南京大学硕士论文铜绿微囊藻群体感应的初步研究撬划一ｈｏｍｏｓｅｒｉｎｅｌａｃｔｏｎｅｓ蹶Ｈ啦”凡轧一八八，觏ＯＨ／＼ＬｕｘＭ缎翱ａ删／＼Ｒｈ！｝艘ａｅｒｕｇｍｏｓａ）０．八．。

／＼八／＼，：人Ｌａｓｉｐ．鑫｛８ｒ甜露挣粥。

ｓ帮Ｏｉｉｇｏｐｅｐｔｉｄｅａｕｔｏｉｎｄｕｃｅｒｓ触祭，斜‰ｅ∥≮拿专毋强ｆ孙ｒ毡～０参ＩＰ－Ｉ￥．ａｕｒｅｕｓ）豁，融、ｔ蝴鲰ｖ。

，您镭，≮～≯。

ｂＡｌＰ—ｔｌ蕊ａｕｒｅｕｓ）Ａ８ｐ，魄－㈣孙一掰～埯酾最一≯矿斌一眇％毯群融ｌｌｌ藩；ａｕｒｅｏｓ）ＡＩＰｑＶ零，薅渊粼菇图卜１革兰氏阴（左）／阳（右）性菌群体感应信号分子结构（ＷａｔｅｒｓａｎｄＢａｓｓｌｅｒ，２００５）革兰氏阴性细菌（Ｇ。

）的群体感应系统是由细菌自身产生的一类信号分子ＡＨＬｓ调节的。

这类信号分子可以自由进出细菌的细胞膜，并在周围环境中积聚，当环境中信号分子累积到一定浓度阈值时，信号分子与特定的受体蛋白Ｒ的Ｎ端结合，形成一定的构象，使Ｒ受体蛋白的Ｃ端能与特定ＤＮＡ序列相结合（目的基因的启动子序列），从而促进或阻碍某些功能基因的表达（ＳｃｈａｕｄｅｒＢａｓｓｌｅｒ２００１）。

同时，信号分子与Ｒ受体蛋白相结合的复合体对ＡＨＬ类信号分子本身及受体蛋白Ｒ的合成具有反馈调节效应（图１．２左）（Ｘａｖｉｅｒ２００３；Ｗａｔｅｒｓ２００５）。

革兰氏阳性细菌（Ｇ＋）群体感应系统不同于革兰氏阴性细菌，调控的信号分子是经修饰过的寡肽，寡肽不能自由穿透细胞膜，需要ＡＢＣ转运系统（ＡＴＰ．Ｂｉｎｄｉｎｇ．ｃａｓｓｅｔｔｅ）或其它膜通道蛋白作用到达胞外行使功能。

寡肽信号随菌体浓度的增加而增加，当累积达到一定浓度阈值时，作用于膜上的信号识别系统（Ｂａｓｓｌｅｒ２００２）。

该识别系统与Ｇ‘细菌中的ＬｕｘＲ类受体蛋白不同，是由双组分磷酸激酶组成的。

膜上激酶与信号分子识别，可以促进激酶中组氨酸残基磷酸化，经过天冬氨酸残基的传递，最终把磷酸基团传递给受体蛋白。

基于群体感应调控细菌生物膜形成研究进展

第２期收稿日期：２０２０－１０－２３作者简介：孙长龙（１９９５—），辽宁鞍山人，硕士，研究方向：污染生态；通信作者：张　阳（１９７５—），女，教授，博士研究生，研究方向：微生物生态。

基于群体感应调控细菌生物膜形成研究进展孙长龙，吴　思，张倩楠，马　信，齐笑萱，张　阳（沈阳师范大学生命科学学院，辽宁沈阳　１１００３４）摘要：近些年来，许多研究都表明细菌等微生物之间存在群体感应现象（ｑｕｏｒｕｍｓｅｎｓｉｎｇ，ＱＳ）。

许多细菌都能合成并分泌自诱导物质来调控基因的表达调控生物膜的形成，以此保证细菌在生长过程中适应新的环境。

生物膜（ｂｉｏｆｉｌｍ）的存在与细菌的抗逆能力有很大的关联，基于驱散群体感应抑制生物膜的形成也成了目前的研究热点之一。

本文主要综述了近几年在群体感应领域的一些发现，主要对细菌生物膜形成相关研究做了一些总结。

关键词：群体感应；生物膜；群体感应抑制剂；细菌中图分类号：Ｓ８５３．７４文献标识码：Ａ文章编号：１００８－０２１Ｘ（２０２０）０２－００８９－０３ＡｄｖａｎｃｅｓｉｎｔｈｅＲｅｇｕｌａｔｉｏｎｏｆＢａｃｔｅｒｉａｌＢｉｏｆｉｌｍＦｏｒｍａｔｉｏｎＢａｓｅｄｏｎＱｕｏｒｕｍＳｅｎｓｉｎｇＳｕｎＣｈａｎｇｌｏｎｇ，ＷｕＳｉ，ＺｈａｎｇＱｉａｎｎａｎ，ＭａＸｉｎ，ＱｉＸｉａｏｘｕａｎ，ＺｈａｎｇＹａｎｇ（ＣｏｌｌｅｇｅｏｆＬｉｆｅＳｃｉｅｎｃｅｓ，ＳｈｅｎｙａｎｇＮｏｒｍａｌＵｎｉｖｅｒｓｉｔｙ，Ｓｈｅｎｙａｎｇ　１１００３４，Ｃｈｉｎａ）Ａｂｓｔｒａｃｔ：Ｉｎｒｅｃｅｎｔｙｅａｒｓ，ｍａｎｙｓｔｕｄｉｅｓｈａｖｅｓｈｏｗｎｔｈａｔｔｈｅｒｅｉｓｑｕｏｒｕｍｓｅｎｓｉｎｇｂｅｔｗｅｅｎｍｉｃｒｏｂｅｓｓｕｃｈａｓｂａｃｔｅｒｉａ（ｑｕｏｒｕｍｓｅｎｓｉｎｇ，ＱＳ）．Ｍａｎｙｂａｃｔｅｒｉａｃａｎｓｙｎｔｈｅｓｉｚｅａｎｄｓｅｃｒｅｔｅｓｅｌｆ－ｉｎｄｕｃｉｎｇｓｕｂｓｔａｎｃｅｓｔｏｒｅｇｕｌａｔｅｇｅｎｅｅｘｐｒｅｓｓｉｏｎａｎｄｒｅｇｕｌａｔｅｂｉｏｆｉｌｍｆｏｒｍａｔｉｏｎ，ｓｏａｓｔｏｅｎｓｕｒｅｔｈａｔｂａｃｔｅｒｉａａｄａｐｔｔｏｎｅｗｅｎｖｉｒｏｎｍｅｎｔｄｕｒｉｎｇｇｒｏｗｔｈ．Ｂｉｏｆｉｌｍ（ｂｉｏｆｉｌｍ）ｈａｓａｇｒｅａｔｒｅｌａｔｉｏｎｓｈｉｐｗｉｔｈｔｈｅｒｅｓｉｓｔａｎｃｅｏｆｂａｃｔｅｒｉａ，ａｎｄｔｈｅｉｎｈｉｂｉｔｉｏｎｏｆｂｉｏｆｉｌｍｆｏｒｍａｔｉｏｎｂａｓｅｄｏｎｄｉｓｐｅｒｓａｌｑｕｏｒｕｍｓｅｎｓｉｎｇｈａｓｂｅｃｏｍｅｏｎｅｏｆｔｈｅｃｕｒｒｅｎｔｒｅｓｅａｒｃｈｈｏｔｓｐｏｔｓ．Ｉｎｔｈｉｓｐａｐｅｒ，ｓｏｍｅｆｉｎｄｉｎｇｓｉｎｔｈｅｆｉｅｌｄｏｆｑｕｏｒｕｍｓｅｎｓｉｎｇｉｎｒｅｃｅｎｔｙｅａｒｓａｒｅｒｅｖｉｅｗｅｄ，ｍａｉｎｌｙｒｅｌａｔｅｄｔｏｔｈｅｆｏｒｍａｔｉｏｎｏｆｂａｃｔｅｒｉａｌｂｉｏｆｉｌｍｍａｄｅｓｏｍｅｓｕｍｍａｒｙ．Ｋｅｙｗｏｒｄｓ：ｑｕｏｒｕｍｓｅｎｓｉｎｇ；ｂｉｏｆｉｌｍ；ｑｕｏｒｕｍｓｅｎｓｉｎｇｉｎｈｉｂｉｔｏｒ；ｂａｃｔｅｒｉａ１　群体感应系统与细菌生物膜的形成１．１　细菌中的群体感应群体感应（ｑｕｏｒｕｍｓｅｎｓｉｎｇ，ＱＳ）又称细胞交流，是指菌体自身产生化学信号并且感知其相应信号浓度变化，进行微生物种间或种内信息交流，从而调节微生物群体行为的一种特殊调控系统。

细菌群体感应调节系统的研究-周纯华

细菌群体感应调节系统的研究兽医学院2010级动物丁颖班周纯华201030710330摘要：细菌与细菌之间的信息交流是通过相互交换一种自动诱导物(autoinducer)的信号分子来实现的。

这种信息交换的过程被称为群体感应(quorum system)。

细菌根据这种特定信号分子浓度的变化来监测环境中其它细菌数量的变化。

细菌的群体感应系统分为种内和种间信息交流两大类。

细菌间的信息交流涉及到细菌的多种生理功能,如细菌的致病能力等。

因此研究细菌间的信息交流有可能找到一条新的防治细菌感染途径。

[1]关键词:群体感应；N-酰基高丝氨酸内酯(AHL)；多肽(AIP)；呋喃酰硼酸二酯(AI)长期以来人们一直以为只有多细胞生物中存在细胞与细胞之间细信息交流，而细菌往往被认为以单纯的单个细胞的生存方式存在于环境中。

然而进入20世纪90年代以后，人们认识到细胞间信息交流不限于真核生物，在原核生物如革兰氏细菌中同样存在着细胞与细胞之间的信息交流。

细菌根据这些信号分子的浓度监测周围环境中自身或其他细菌的数量变化，并通过信号分子发出信号，启动菌体中相关基因的表达，改变和协调它们之间的行为，共同展示出它们的某些生理特性，从而表现出单个细菌无法从事的某些生理功能和调节机制。

这一调控系统被称为细菌的群体感应调节（quorum sensing,QS）。

今年研究证实，QS参与细菌的多种生理行为和生物学功能的调控，包括菌体生物发光，浮游，生物膜形成，抗生素合成，病原致病因子产生等。

[2]1.细菌种内特异性的群体感应系统1.1革兰氏阳性细菌的群体感应系统革兰氏阳性细菌主要以多肽(autoinducing polypeptides,简称AIP)为信号分子。

[3]所有的AIP分子都是在细胞质中由前导肽切割、加工而成为成熟的信号分子。

AIP分子的氨基酸残基一般为8～9,C端第5位是一个保守的半胱氨酸,它与C末端的氨基酸残基以硫酯键相连,形成类酯。

相关主题

1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。

南京农业大学学报　2009,32(3):7177Journal of Nanjing Agricultural University 谷春艳,范加勤,杨雪,等.彩色马蹄莲细菌性软腐病菌的鉴定及其群体感应淬灭的研究[J].南京农业大学学报,2009,32(3): 7177彩色马蹄莲细菌性软腐病菌的鉴定及其群体感应淬灭的研究谷春艳1,范加勤1*,杨雪1,胡白石1,刘凤权1,张永春2(1.南京农业大学植物保护学院/农业部病虫监测与治理重点开放实验室,江苏南京210095;2.上海市农业科学院花卉研究中心,上海201106)摘要:近几年南京种植的彩色马蹄莲软腐病发生严重,从不同品种的彩色马蹄莲不同部位病组织中分离到6个菌株,经形态特征与培养性状比较㊁生理生化测试㊁16S rDNA序列分析和致病性测定,鉴定为胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovora subsp.carotovora,P.c.c)㊂信号分子检测结果表明,分离菌株可产生并释放出N酰基高丝氨酸内酯(AHLs)类信号分子㊂利用PCR技术扩增土壤根癌农杆菌中的attM基因,其编码的AttM解酯酶可以显著降解AHLs,在离体条件下可有效地减弱该病原菌的致病性,这为有效控制彩色马蹄莲细菌性软腐病提供了一种新途径㊂关键词:彩色马蹄莲;细菌性软腐病;病原菌鉴定;attM;群体感应淬灭中图分类号:S682.2+64;S432.4+2 文献标志码:A 文章编号:10002030(2009)03007107 Identification of the pathogen and quorum quenching studyon bacterial soft rot of colored calla lilyGU Chun⁃yan1,FAN Jia⁃qin1*,YANG Xue1,HU Bai⁃shi1,LIU Feng⁃quan1,ZHANG Yong⁃chun2(1.College of Plant Protection/Key Laboratory of Monitoring and Management of Plant Diseasesand Insects,Ministry of Agriculture,Nanjing Agricultural University,Nanjing210095,China;2.Flowers Research Center,Shanghai Academy of Agricultural Sciences,Shanghai201106,China) Abstract:Bacterial soft rot occurred seriously in colored calla lily(Zantedeschia spp.)in Nanjing,China.Six isolates from dif⁃ferent parts of rotted calla lily were identified as Pectobacterium carotovora subsp.carotovora by morphological observation,bio⁃chemical and physiological characteristics,16S rDNA sequences analysis and pathogenicity determination.The results showed that the isolated strains produced N⁃acyl homosrine lactones(AHLs)detected by AHL⁃bioassay strain JZA1.Furthermore,the lacto⁃nase AttM coded by gene attM amplification from Agrobacterium tumefaciens could degrade AHLs effectively.Suppression of calla lily soft rot was observed after the plants were inoculated with the mixture of the pathogen and AttM,which provided a promising means to control the bacterial soft rot disease of colored calla lily.Key words:colored calla lily;bacterial soft rot disease;pathogen identification;attM;quorum quenching20世纪90年代初,全球性的市场需求促进了彩色马蹄莲(Zantedeschia spp.)的产业形成,并使其成为具有发展潜力的球根花卉品种㊂但由于细菌性软腐病发生严重,且没有理想的防治对策,极大阻碍了彩色马蹄莲的发展[1-3]㊂20世纪末,中国开始引种彩色马蹄莲[4];易建平等[5]报道了进口马蹄莲海关抽样材料中存在软腐病㊂随着组培产业化技术的应用[6],国内种植规模不断增加,软腐病发生严重时达100%;王敏等[7]报道云南马蹄莲软腐病病原菌中,除Pectobacterium carotovora subsp.carotovora 外,还有P.chrysanthemi;而彩色马蹄莲病害防控方面的研究报道则较少[8]㊂细菌不同细胞间存在化学信号调节,当群体达到一定密度时,该信号分子达到一定浓度,进而启动控制各种生物行为的基因(包括致病基因)表达,即群体感应(quorum sensing)[9]㊂针对P.carotovora subsp.carotovora的信号分子(AHL)㊁作用方式和群体感应淬灭(quorum quenching)已有较多的研究收稿日期:20080401基金项目:江苏省农业三项工程项目(SX(2002)058);江苏省科技兴农项目(BC2001357);上海市农业科技攻关项目(沪农科攻字(2007)第12号)作者简介:谷春艳,硕士研究生㊂*通讯作者:范加勤,从事植物病理学研究,E⁃mail:fanjq@㊂报道[9-12]㊂Dong 等[9-10]证实源于Bacillus sp.的aiiA 基因产生的解酯酶AiiA 可降解马铃薯软腐病菌的群体感应信号分子AHL;Molina 等[13]提出解酯酶AiiA 不但可以防治软腐病的发生,而且具有一定的治疗效果㊂attM 基因与aiiA 基因相似,Zhang 等[14]和Dong 等[15]对其干扰细菌群体感应进行了报道,但国内尚无有关的研究报道㊂本研究以南京栽培彩色马蹄莲软腐病为研究对象,对病原菌进行分离鉴定㊂利用PCR 扩增,从土壤根癌农杆菌(Agrobacterium tumefaciens )中获得attM 基因,通过病原菌群体感应信号分子AHL 的生物检测,以及attM 基因和aiiA 基因对病原菌致病性抑制作用比较,为进一步利用attM 基因防控彩色马蹄莲细菌性软腐病奠定基础㊂1　材料与方法1.1　供试菌株及来源本研究所用菌株与质粒及其特性见表1㊂表1　供试菌株和质粒Table 1　Strains and plasmids used in this study 菌株/质粒Strain /plasmid相关特性Relevant characteristics 来源Reference /source 菌株Strain 胡萝卜软腐果胶杆菌P .carotovora 初次分离菌株(PccS)Isolate from rotted tissue Wild type 本研究This study 回接分离菌株(RPccS)Isolate from rotted tissue inoculated with PccS Wild type,isolated from the rotted plant cultured in greenhouse 本研究This study 胡萝卜软腐果胶杆菌胡萝卜亚种 Pectobacterium carotovora subsp.carotovora (P .c .c )Wild type,the standard strain 本研究室This laboratory 大肠杆菌E .coli DH5αΦ90lacZ Δm 15,recA 1本研究室This laboratory DH5α/pME6000DH5αtransformant with pME6000plasmid 本研究This study DH5α/pME6863DH5αtransformant with pME6863plasmid,AHL degrader 本研究This study DH5α/pUCM DH5αtransformant with pUCM plasmid,AHL degrader 本研究This study DH5α/pUC19DH5αtransformant with pUC19plasmid 本研究This study 土壤根癌农杆菌Agrobacterium tumefaciens本研究室This laboratory JZA1AHL biosensor strain KYC55(pJZ372)[Tc R ](pJZ384)[Spe R ](pJZ410)[Gm R ][11] R10(pCF 218)Ptet⁃traR,Tc R ,high AHL production strain [12]质粒Plasmids pME6000Broad⁃host⁃range cloning vector,Tc R [16]　pME6863pME6000carrying the aiiA gene from Bacillus sp.A24under the constitutive Plac promoter [17]　pUC19Cloning vector,Amp R 本研究室This laboratory　pUCM pUC19carrying the attM gene from A .tumefaciens EHA105本研究This study 1.2　菌株分离将来自种植圃或回接发病的病株组织表面消毒后采用平板划线分离法[18],在肉汁胨(NA)平板培养基上培养分离纯化3次,-20℃保存备用㊂1.3　细菌形态特征观察及生理生化测试1.3.1　细菌染色试验　按常规方法采用结晶紫㊁草酸铵染色法[19],赖夫生鞭毛染色法和西萨基尔染色法[18]进行染色㊁镜检㊂1.3.2　生理生化测试　参照Krieg 等[20]和Duveiller 等[21]的方法进行氧化酶㊁接触酶反应,测定淀粉水解㊁好氧性和厌氧性;蛋白胨葡萄糖磷酸盐培养[19],进行甲基红(MR)和V.P.试验;常规方法[18]观察H 2S 变黑反应㊁硝酸盐及亚硝酸盐的还原反应㊁利用氨基酸和碳水化合物产酸反应㊂1.4　致病性测定1.4.1　烟草过敏性反应　将28℃培养24h 的菌株,用无菌水配成1×108CFU㊃mL -1的菌悬液,注射普通烟叶背面的表皮下,温室培养24h 后观察㊂27 南　京　农　业　大　学　学　报第32卷1.4.2　彩色马蹄莲致病性测定　分离物在NA 固体培养基上培养24h,用无菌水配制1×108CFU㊃mL -1的菌悬液,注射彩色马蹄莲叶柄背面距土面5cm 处,每株注射10μL,脱脂棉保湿8~12h㊂种球用70%乙醇表面消毒,切成厚约5~6mm 薄片,每片接10μL 菌悬液,以无菌水作为对照,均重复3次,28℃恒温培养,观察发病情况㊂1.4.3　马铃薯㊁大白菜的致病性测定　取新鲜马铃薯块茎和大白菜叶柄洗净,用70%乙醇表面消毒,马铃薯块茎切成厚约5~6mm 薄片,大白菜叶柄切成约30mm ×90mm 的长条㊂每个培养皿放1片马铃薯块茎或1块大白菜叶柄,培养皿中加灭菌水5mL㊂每片接3处,每处接5μL 菌悬液,以无菌水和P .c .c 作对照,均重复3次,28℃培养12h 观察结果㊂1.5　16S rDNA PCR 扩增1.5.1　DNA 提取　分离物按1%体积比接入NA 液体培养基,28℃㊁220r㊃min -1振荡培养20h㊂用购自TIANGEN 的TIANamp Bacteria DNA Kit 提取病菌基因组DNA㊂取3μL DNA 溶液于1%琼脂糖凝胶上电泳,确定PCR 时的模板量㊂1.5.2　PCR 反应体系　10×Buffer (10mmol㊃L -1Tris⁃HCl,pH 8.3,50mmol㊃L -1KCl)2.5μL,25mmol㊃L -1MgCl 21.5μL,2.5mol㊃L -1dNTP 1μL,r Taq polymerase 0.5U (以上试剂均购自大连宝生物有限公司),16S rDNA 特异性引物(20mmol㊃L -1)(Invitrogen 公司合成)各1μL,病菌基因组DNA 1μL,双蒸水补足至25μL㊂PCR 反应程序:95℃5min;94℃30s,54℃30s,72℃2min,30个循环;72℃10min㊂取8μL 扩增产物,1%琼脂糖凝胶电泳分离检测㊂1.5.3　PCR 扩增产物测序　部分PCR 产物,按胶回收试剂盒说明回收纯化,送上海英骏生物技术有限公司测序㊂将测得的序列与GenBank 数据库中已注册的序列用BLAST 搜索软件进行同源性搜索,运用Mega 生物软件进行分析㊂1.6　分离菌株的自诱导物检测经分离鉴定的菌株,参照Zhu 等[11]的生物显影方法检测群体感应自体诱导物AHL,JZA1为检测菌株,R10为阳性对照,NA 液体培养基为阴性对照㊂1.7　pUCM 载体的构建根据已公布的Agrobacterium tumefaciens 中的attM 基因序列(GenBank 登录号U59485,所处的位置15833~16603)设计引物(attmF ∶5′⁃GCCAAGCTTATGCTTCAGTCGGGTACG⁃3′;attmR ∶5′⁃GCCGAAT⁃TCTTACGCGTAAAATTCGGG⁃3′),以A .tumefaciens EHA105的基因组DNA 为模板扩增attM 基因㊂用TaKaRa 公司提供的高效保真酶(code:DR010S)进行PCR 扩增,反应条件:98℃5min;98℃10s,60℃30s,72℃1min,30循环;72℃8min㊂将PCR 产物酶切回收连接于pUC19上,转化宿主菌DH5α,进行PCR 扩增和酶切验证,并送上海英骏生物技术有限公司进行序列测定㊂1.8　解酯酶AttM 对病原菌信号分子降解作用的检测将DH5α/pUCM ㊁DH5α/pUC19分别与AHL 阳性菌株R10上清液,按体积比1∶1共同接种于含有氨苄青霉素(Ampicillin,Amp)的液体LB 培养基中,37℃振荡过夜,各自取菌液和上清液分别利用JZA1菌株进行生物显影㊂以R10为阳性对照,以DH5α/pUC19的菌液和上清液分别作阴性对照㊂用β半乳糖苷酶法[22]检测β半乳糖苷酶活性,重复3次,所得结果用SAS 软件Duncan′s 多重比较法进行统计分析㊂1.9　解酯酶AttM 对彩色马蹄莲软腐病菌菌体生长的影响将DH5α/pUC19㊁DH5α/pUCM 和测序鉴定的彩色马蹄莲软腐病分离物(PccS1)培养12h,利用分光光度计分别将各菌液的D 600调整为0.1㊂将空白LB 培养液㊁DH5α/pUC19㊁DH5α/pUCM 分别与PccS1等体积混合后分别接种到20mL LB 培养液中㊂经30℃过夜培养后利用分光光度计分别将各菌液的D 600再次调整为0.1,立即按梯度稀释法稀释105倍㊂吸取各菌液100μL 分别涂于含有Amp 抗生素和不含抗生素的LB 平板上,每个处理重复3次,30℃培养48h㊂统计含有抗生素和不含抗生素平板的菌落数,按Duncan′s 新复极差法测验各处理间的差异显著性㊂1.10　离体条件下,解酯酶AiiA ㊁AttM 对彩色马蹄莲软腐病菌的致病性影响37℃下,DH5α/pME6863㊁DH5α/pME6000㊁DH5α/pUCM㊁DH5α/pUC19培养12h,PccS128℃下培养12h,均用无菌水配成1×108CFU ㊃mL -1的菌悬液㊂将DH5α/pME6863㊁DH5α/pME6000㊁37第3期谷春艳,等:彩色马蹄莲细菌性软腐病菌的鉴定及其群体感应淬灭的研究DH5α/pUCM㊁DH5α/pUC19的菌悬液分别与病原菌分离物的菌悬液等体积混合,接种于彩色马蹄莲种球切片(厚约5~6mm)㊂每培养皿1片,加灭菌水5mL,每片接种10μL菌悬液混合物,以PccS1和无菌水分别作对照,每个处理重复3次,28℃恒温箱培养12h㊂用十字交叉法计算腐烂面积的大小㊂所得数据按Duncan′s新复极差法测验各处理间的差异显著性㊂2　结果与分析2.1　病菌的培养性状及形态特征从彩色马蹄莲不同品种㊁不同部位的病组织中,获得6个分离物(PccS),NA平板上培养24h后可见直径约1mm的圆形菌落,灰白色,透明,表面湿润光滑稍隆起,后期变为半透明㊂将PccS回接健康植株,再分离发病组织,获得的回接分离物(RPccS)培养性状与PccS一致㊂菌体均为直杆状,单生或串生㊂革兰氏反应阴性,周鞭2~5根㊂2.2　生理生化测定分离的菌株和对照菌株P.c.c均为兼性厌气菌㊂氧化酶㊁淀粉酶反应均为阴性,接触酶阳性㊂发酵产酸,产气,甲基红试验㊁硝酸盐还原反应为阳性,V-P试验为阴性,能利用多种碳水化合物和氨基酸,但分离菌株均不能利用半胱氨酸产生硫化氢,而P.c.c可以利用半胱氨酸产生硫化氢㊂2.3　彩色马蹄莲软腐病菌分离及致病性测定6个分离物,分别接种盆栽彩色马蹄莲健康株,均能引起发病,症状与田间自然症状一致(图1)㊂起初接种处出现水渍状,界限明显,随着病势的发展,病组织开始软化㊁腐烂最后全株倒伏死亡㊂图2显示初次分离菌株PccS㊁回接分离菌株RPccS及对照菌株P.c.c在烟叶背面注射接种,24h后均有过敏性反应㊂图3所示,P.c.c㊁PccS接种马铃薯块茎和彩色马蹄莲种球切片㊁大白菜叶柄均引起软腐, 3种情况下,后者产生的病斑面积均大于前者,对照(接种无菌水)未出现腐烂㊂图1　分离物回接盆栽健康植株症状Fig.1　Potted colored calla lily soft rotted after inoculated with cells collected from pathogen on agar plate图2　烟叶过敏性反应Fig.2　Hypersensitive reaction on tobacco leaf 1.对照菌株P.c.c;2.无菌水H2O;3.初次分离菌株1 PccS1;4.回接分离菌株1RPccS1;5.初次分离菌株2PccS2;6.回接分离菌株2RPccS2图3　分离物及对照菌株P.c.c接种离体植物组织发病症状Fig.3　Plant in vitro inoculation with isolated pathogen and P.c.cA.马铃薯块茎切片Potato tuber slice;B.彩色马蹄莲种球切片Colored calla lily tuber slices;C.大白菜叶柄Cabbage leafstalk1.对照菌株P.c.c;2.初次分离菌株PccS;3.无菌水H2O2.4　16S rDNA PCR扩增及序列测定将6个分离物用16S rDNA的特异性引物进行PCR扩增㊁测序㊂6个分离物的同源性为100%,选47 南　京　农　业　大　学　学　报第32卷其中一个代表菌株PccS1与GenBank 数据库中已注册P .c .c 的序列用BLAST 程序进行同源性比较,结果同源性为99%,由此确定分离的6个菌株均为Pectobacterium carotovora subsp.carotovora ㊂图4为用Mega 生物软件所作的同源性比较聚类图㊂图4　PccS116S rDNA 同源性比较聚类图Fig.4　Clustering for homologic comparison of the PccS116S rDNA2.5　分离菌株的AHL 活性测定图5结果表明,上述6个分离物中的2个代表菌株与R10阳性对照均能产生显色反应,而NA 液体培养基不能㊂表明彩色马蹄莲软腐病病原菌能够产生并释放出AHL 信号分子㊂2.6　AttM 解酯酶对AHL 信号分子的降解作用从图6可以看出,载体DH5α/pUC19与R10共同培养的菌液及上清液能够产生显色反应,而DH5α/pUCM 与R10共同培养的菌液及上清液显色反应很弱,β半乳糖苷酶活性明显降低,均不足阳性对照的1/6㊂由此可见,DH5α在导入attM 基因后能够显著降解R10产生的AHL 信号分子㊂图5　分离菌株的AHL 活性测定Fig.5　AHL activation of the isolated pathogen 1.R10;2.PccS1;3.PccS2;4.NA图6　AttM 解酯酶对AHL 信号分子的降解作用Fig.6　Degradation effects of AttM on AHL左图:AHL 生物显色反应;右图:β半乳糖苷酶活性㊂Left:AHL⁃bioassay strain JZA1;Right:Activities of β⁃galactosidase.1.R10;2.DH5α/pUCM+R10(上清液Supernatant);3.DH5α/pUCM +R10(菌液Mixture);4.DH5α/pUC19(上清液Superna⁃tant);5.DH5α/pUC19(菌体悬浮Bacteria);6.DH5α/pUC19+R10(上清液Supernatant);7.DH5α/pUC19+R10(菌液Mixture)不同大写字母表示邓肯氏检验1%水平上差异显著㊂The different capital letters show the significance at 0.01level using Duncan′s test.The same as follows.2.7　解酯酶AttM 对彩色马蹄莲软腐病菌菌体生长的影响由图7显示,DH5α/pUC19和DH5α/pUCM 与PccS1混合培养,均对PccS1生长存在抑制作用,但57第3期谷春艳,等:彩色马蹄莲细菌性软腐病菌的鉴定及其群体感应淬灭的研究图7　AttM对PccS1菌体生长的影响Fig.7　Effects of AttM on the cell growth of PccS1 1.PccS1;2.DH5α/pUC19+PccS1;3.DH5α/pUCM+PccS1两者之间差异不显著㊂表明DH5α/pUC19和DH5α/pUCM对彩色马蹄莲细菌性软腐病菌PccS1生长的影响与解酯酶AttM关系不显著㊂2.8　AttM解酯酶对彩色马蹄莲软腐病菌致病性的抑制如图8所示,分离物PccS1接种彩色马蹄莲种球切片产生腐烂面积最大,其与等体积DH5α/ pME6000㊁DH5α/pUC19混合接种,腐烂面积均稍有减小,与DH5α/pME6863㊁DH5α/pUCM共同接种,腐烂面积显著减小;无菌水接种则不发病㊂表明DH5α/pUCM中的attM基因编码的产物,与DH5α/pME6863中的aiiA基因编码的产物对P.carotovora subsp.carotovora引起的彩色马蹄莲软腐病具有类似的防治效果㊂图8　AiiA和AttM对彩色马蹄莲软腐病菌致病性抑制Fig.8　The control effects of AiiA and AttM on colored calla lily soft rot caused by P.carotovora subsp.carotovora 左图Left:上排从左到右The top row left to right:PccS1,DH5α/pME6000+PccS1,DH5α/pME6863+PccS1下排从左到右The bottom row left to right:H2O,DH5α/pUC19+PccS1,DH5α/pUCM+PccS1,respectively 右图Right:1.PccS1;2.DH5α/pME6000+PccS1;3.DH5α/pME6863+PccS1;4.DH5α/pUC19+PccS1;5.DH5α/pUCM+PccS1;6.H2O不同小写字母表示经邓肯氏检验在5%水平上差异显著㊂The different small letters show the significance at0.05level usingDuncan′s test.3　讨论本研究表明,根据分离物的形态特征㊁培养性状㊁生理生化特性㊁致病性和16S rDNA测序结果,南京地区彩色马蹄莲软腐病的病原菌被鉴定为胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovo⁃ra subsp.carotovora,P.c.c),与多数研究结果一致[1-3,5],排除了P.chrysanthemi同是病原菌[7]的可能㊂在几种情况下,分离物的致病力比标准菌株P.c.c强,这可能与保存菌株的致病性下降有关[16]㊂生物显色反应表明:1)自彩色马蹄莲细菌性软腐病组织中分离获得的菌株能够产生AHL;2)自A.tumefaciens中获得的attM基因,能在DH5α中有效地表达并产生AHL降解酶(AHL⁃lactonase),与有关文献报道结果一致[14-15]㊂attM基因编码的解酯酶AttM对彩色马蹄莲软腐病菌菌体生长和致病性的测定试验结果证实,解酯酶AttM不影响彩色马蹄莲软腐病菌的菌体生长,但可以通过降解AHLs影响软腐病菌的致病性,这与当前国际植物病理界研究热点的aiiA基因[9-10]具有相似功能,为进一步对彩色马蹄莲细菌性病害防治开创了良好的理论基础㊂本试验中DH5α/pUC19和DH5α/pUCM与PccS1混合物中PccS1生长量降低,可能与2种微生物在同一环境中的营养竞争有关㊂因本研究中使用的表达载体是pUC19,启动子为lac,其活性相对较弱,可能会影响attM基因的表达量,且在表达过程中需要IPTG诱导,如能采用强启动子,如Plpp等,可能会取得更好的效果㊂后续工作可通过转基因技术,将attM基因导入彩色马蹄莲或构建生物防治基因工程菌,可有效防控彩色马蹄莲细菌性软腐病,因为利用aiiA基因在这两方面研究已取得了令人兴奋的结果[9-13]㊂参考文献:[1]　陈俊位,林俊义.彩色海芋细菌性软腐病防治方法之探讨[J].植物病理学会刊,2000,9(3):10711467 南　京　农　业　大　学　学　报第32卷[2]　Snijder R C,Cho H R,Hendriks M M W B,et al.Genetic variation in Zantedeschia spp.(Araceae )for resistance to soft rot caused byErwinia carotovora subsp.carotovora [J].Euphytica,2004,135:119128[3]　李世峰.是什么制约了彩色马蹄莲切花生产[J].中国花卉园艺,2001(13):1617[4]　王爱红,吴彩琼,贾彩娟,等.彩色马蹄莲引种栽培实验[J].广东园林,2006,28(2):3942[5]　易建平,戚龙君,陶庭典,等.马蹄莲细菌性软腐病菌的鉴定[J].植物检疫,2002,16(1):810[6]　范加勤,张雯雯,张娜,等.几个彩色马蹄莲品种的离体培养与快速繁殖[J].南京农业大学学报,2005,28(2):2831[7]　王敏,姬广海,修建华,等.云南省马蹄莲细菌性软腐病原鉴定[J].西南大学学报:自然科学版,2007,29(8):7982[8]　赵培飞,吴丽芳,郑凌,等.云南彩色马蹄莲的生产及发展前景[J].湖南农业科学,2002(3):6970[9]　Dong Y H,Wang L H,Xu J L,et al.Quenching quorum sensing dependent bacterial infection by an N ⁃acyl homoserine lactonase [J].Nature,2001,411:813817[10]Dong Y H,Xu J L,Li X Z,et al.AiiA,an enzyme that inactivates the acylhomoserine lactone quorum⁃sensing signal and attenuates the virulence of Erwinia carotovora [J].Proc Natl Acad Sci USA,2000,97:35263531[11]Zhu J,Chai Y,Zhong Z,et al.Agrobacterium bioassay strain for ultrasensitive detection of N ⁃acylhomoserine lactone⁃type quorum⁃sensing molecules:detection of autoinducers in Mesorhizobium huakuii [J].Appl Environ Microbiol,2003,69:69496953[12]Fuqua W C,Winans S C.A LuxR⁃LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite [J].J Bacteriol,1994,176:27962806[13]Molina L,Constantinescu F,Michel L,et al.Degradation of pathogen quorum⁃sensing molecules by soil bacteria:a preventive and curative biological control mechanism [J].FEMS Microbiol Ecol,2003,45(1):7181[14]Zhang H B,Wang L H,Zhang L H.Genetic control of quorum⁃sensing signal turnover in Agrobacterium tumefaciens [J].Proc Natl Acad Sci USA,2002,99(7):46384643[15]Dong Y H,Zhang L H.Quorum sensing and quorum⁃quenching enzymes [J].The Journal of Microbiology,2005,43:101109[16]Maurhofer M,Reimmann C,Schmidli⁃Sacherer P,et al.Salicylic acid biosynthetic genes expressed in Pseudomonas fluorescens strain P3improve the induction of systemic resistance in tobacco against tobacco necrosis virus [J].Phytopathology,1998,88:678684[17]Reimmann C,Ginet N,Michel L,et al.Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1[J].Microbiology,2002,148:923932[18]方中达.植病研究方法[M].3版.北京:中国农业出版社,1998[19]任欣正.植物病原细菌的分类和鉴定[M].北京:农业出版社,1994[20]Krieg N R,Holt J G.Bergey′s Manual of Systematic BacteriologyⅠ[M].Baltimore:Williams and Wilkins,1984[21]Duveiller E,Bragard C,Rudolph K,et al.The Bacterial Diseases of Wheat:Concepts and Methods of Disease Management [M].Mexico D F:CIMMYT,1997[22]高轶静,钟增涛,郑会明,等.华癸根瘤菌中自体诱导物的初步研究[J].微生物学报,2005,45(1):1922责任编辑:夏爱红77第3期谷春艳,等:彩色马蹄莲细菌性软腐病菌的鉴定及其群体感应淬灭的研究。