【作者】 郭秀春；

【导师】 王小如； 郑立；

【作者基本信息】 中国海洋大学 ， 海洋化学， 2011， 博士

【摘要】 由于复杂和特殊的生存环境,海洋微生物在海洋环境中通常与其他生物形成共附生的关系,并形成一套化学防御系统以利于自身或宿主的生存,而所形成的化学防御物质大多具有生物活性,所以越来越多的科学家认为化学防御是海洋微生物产生活性物质的主要诱因。但海洋微生物化学防御是如何启动,即它的启动机制是什么,还鲜有研究。随着微生物学的发展,群体感应机制逐渐被揭示,有学者提出微生物,尤其是细菌中的化学防御机制是受到群体感应调控的假设。群体感应(quorum sensing)是细菌细胞根据自身和外源细菌所释放的特定信号分子浓度,以感知细胞数量变化,并进行基因表达的代谢调控现象。群体感应最重要的一个特点是密度依赖性,即当细菌密度较高时,细胞产生的自诱导信号分子(AI)浓度会积累到一个临界阈值,细菌感应到自诱导信号分子,从而表现出某些独特的生理特性,包括生物发光、抗生素的生物合成、毒性基因的表达及生物膜的形成等等。本论文所要解决的问题是探明海洋细菌化学防御物质的产生是否受到群体感应的调控。本文通过天然产物化学和化学生态学的方法,以一株具有显著抗菌活性且具有群体感应现象的海绵共栖细菌Pseudoalteromonas sp. NJ6-3-1为个案,以产生抗菌物质作为化学防御的响应,一方面鉴定了细菌NJ6-3-1产生的化学防御性物质,并证实细菌NJ6-3-1抗菌物质的产生具有密度依赖性,即抗菌物质的产生受到群体感应的调控,以此说明海绵共栖细菌NJ6-3-1存在由群体感应调控的化学防御;另一方面探明了海绵共栖细菌NJ6-3-1中存在着群体感应信号分子N-酰基高丝氨酸内酯类化合物(AHLs)和二酮哌嗪类化合物(DKPs),并对细菌NJ6-3-1中调控抗菌物质代谢的自诱导信号分子进行了探寻。最后通过对细菌NJ6-3-1进行全基因组测序的方法,在细菌NJ6-3-1中获得了群体感应转录调控蛋白LuxR的基因片段,从而从分子水平上初步证实了细菌NJ6-3-1中确实存在LuxI/LuxR调控的群体感应系统。本项研究将丰富海洋细菌化学防御诱导机制的相关理论,进一步加深对细菌群体感应调控功能的认识,并为开发新型药用天然产物提供新的思路。全文共分六章:第一章,分为两部分,首先介绍了海绵共栖微生物的研究概述,包括海绵共栖微生物的种类、次级代谢产物和化学防御机制;然后介绍了细菌群体感应系统的研究概况,包括群体感应的研究历史、目前发现的群体感应信息系统及信号分子的种类、群体感应系统的形成和调控机制及群体感应信号分子AHLs的检测方法、从而引出海洋细菌中的群体感应现象;在此基础上确定了本文的主要研究内容。第二章,主要研究了具有抗菌活性的海绵共栖细菌的筛选及鉴定。首先利用平板涂布法从南海和东海海绵生物样品中分离海绵共栖细菌,以4种致病菌作为实验用指示菌对海绵共栖细菌进行了抗菌活性筛选。同时采用16S rDNA同源性和系统发育分析对活性菌株进行种属鉴定和系统发生学研究。结果表明,在分离到的95株海绵共栖细菌中,19株(20 %)细菌具有抗菌活性。并且通过细菌分类鉴定,结果显示具有抗菌活性的细菌大部分属于芽孢杆菌属(Bacillus)。第三章,主要研究了海绵共栖细菌Pseudoalteromonas sp.NJ6-3-1抗菌物质产生的密度依赖性。本章第一节和第二节采用化学方法和生物技术相结合的方法,首先对具有显著抗菌活性的海绵共栖细菌NJ6-3-1的抗菌代谢产物进行分离纯化,通过活性跟踪,最终从细菌NJ6-3-1代谢产物中获得了2个抗菌物质,并结合各种结构鉴定的方法确定了化合物的结构。第一个抗菌物质分子式为C15H13BrO3,分子量为320,结构式命名为:2-(1’-溴-1’,3’-丁二烯)-4-(2”,4”-二烯戊酸)-苯酚。该化合物对枯草芽孢杆菌(BS)、金黄色葡萄球菌(SA)、酿酒酵母(SC)均有不同程度的抑菌作用,MIC分别为500μg/mL,125μg/mL和250μg/mL。第二个抗菌化合物分子量为520,分子式为C23H24BrN2SO5。该该化合物对金黄色葡萄球菌(SA)有抑菌作用,MIC为500μg/mL。本章第三节利用酶标仪和高效液相色谱及飞行时间质谱仪同时检测不同培养时间下细菌NJ6-3-1的细胞密度和NJ6-3-1代谢产物粗提液中抗菌物质C23H24BrN2SO5的量,从而确定了细菌NJ6-3-1抗菌物质C23H24BrN2SO5的量依赖于细菌的细胞密度。通过对不同培养时间下细菌NJ6-3-1代谢产物粗提液的抗菌活性检测,进一步证实了细菌NJ6-3-1抗菌物质的代谢具有密度依赖性,且细胞密度阈值为OD630=0.4。第四章,主要研究了海绵共栖细菌Pseudoalteromonas sp.NJ6-3-1代谢产物中的AHLs。本章第一节建立了一套检测细菌中群体感应信号分子AHLs的方法,并结合这些方法对海绵共栖细菌NJ6-3-1中AHLs的产生情况进行了检测。首先通过微生物传感菌A. tumefaciens A136检测,确定细菌NJ6-3-1中含有AHLs信号分子,能够诱导A136产生蓝色色素。再通过GC-MS检测确定细菌NJ6-3-1中含有N-己酰基高丝氨酸内酯(C6-HSL)、N-辛酰基高丝氨酸内酯(C8-HSL)和N-十四酰基高丝氨酸内酯(C14-HSL)三种AHLs信号分子。该方法还可用于大批量细菌中AHLs产生菌的筛选工作。本章第二节通过向低密度细菌NJ6-3-1中添加作为群体感应诱导信号分子的NJ6-3-1的自身代谢物C6-HSL、C8-HSL和C14-HSL的甲醇溶液,获得了添加不同诱导物后的NJ6-3-1代谢产物粗提液,通过生物自显影检测抗菌活性及HPLC检测抗菌物质C23H24BrN2SO5的代谢量,结果初步证实了C8-HSL能够诱导低密度细菌NJ6-3-1产生抗菌活性,并且能够诱导低密度细菌NJ6-3-1的抗菌物质C23H24BrN2SO5代谢量增加,由此我们推断C8-HSL很有可能就是细菌NJ6-3-1产生的调控抗菌物质代谢的自诱导信号分子。第五章,主要研究了海绵共栖细菌Pseudoalteromonas sp.NJ6-3-1代谢产物中的DKPs。首先通过向低密度细菌NJ6-3-1中添加可作为群体感应机制诱导信号分子的NJ6-3-1自身代谢物cyclo-(ΔVal-L-Val)、cyclo-(L-Phe-L-Val)、cyclo-(L -Pro-L-Leu) (简称为VV-2、VF-2、PL-2),获得了添加不同诱导物后的细菌NJ6-3-1代谢产物粗提液,通过生物自显影检测抗菌活性,结果初步证实VF-2能诱导低密度细菌NJ6-3-1产生抗菌活性,因此很有可能就是细菌NJ6-3-1产生的调控抗菌物质代谢的自诱导信号分子。其次,为了从海绵共栖细菌NJ6-3-1中得到更多的DKPs化合物,我们发酵100 L细菌NJ6-3-1菌液,并对细菌NJ6-3-1代谢产物中的DKPs化合物进行分离纯化,共得到12种DKPs化合物,后来通过理化性质及光谱学手段鉴定了DKPs的结构。并且其中1种DKPs化合物,即cyclo-(Tyr-Phe)至今还没有在微生物中发现的报道。第六章,初步研究了海绵共栖细菌Pseudoalteromonas sp.NJ6-3-1中群体感应的分子基础,对luxR基因片段进行了测序分析。通过对细菌NJ6-3-1进行SOLiD全基因组测序的方法(宁波大学工作),在细菌NJ6-3-1中获得了群体感应转录调控蛋白LuxR的基因片段,从而从分子水平上初步证实了细菌NJ6-3-1中存在LuxI/LuxR调控的群体感应系统。更多还原

【Abstract】 Owing to the complex and special living circumstance they reside, marine microorganisms usually coexist in the marine environment, and they gradually form a set of chemical defense system that is beneficial to the survival of themselves or the host. Most of the chemical defense substances they produce have biological activity, so more and more scientists considered that chemical defense mechanism is the main inducing factor for marine microorganisms to produce bioactive substances. However, there are few people studying about how chemical defense start by marine microorganisms and what is starting mechanism. Quorum sensing phenomenon has been gradually revealed with the development of microbiology, and some scholars proposed the hypothesis that chemical defense mechanism in microorganism, especially bacteria, is regulated by quorum sensing.Quorum sensing is a metabolic regulation and control process which enables the bacterial cells to sense changes in cell density and induce the expression of specific genes. This regulatory process is mediated through sensing the concentration changes of specific signal molecules being released either by bacteria themselves, or from external sources. Density-dependent is one of the most important characteristics of quorum sensing, that is, when the bacteria grow to a high cell density, the concentration of autoinducers (AI) they release could reach a critical threshold value. The bacteria could then sense the autoinducers and appear some specific physiological characters, including bioluminescence, the production of antibiotics, virulence genes expression, biofilm formation and so on. In this thesis, we have to solve the problem whether the production of chemical defense substances is regulated by quorum sensing in marine bacteria.Based on natural product chemistry and chemical ecology approach, a sponge-associated bacterium Pseudoalteromonas sp. NJ6-3-1 with remarkable biological activities and with the phenomenon of quorum sensing was chosen as case study, and the production of antibacterial substances was taken as a chemical defense response in this dissertation. On the one hand, the chemical defense substances produced by bacteria NJ6-3-1 was identified, and the production of antibacterial substances of bacteria NJ6-3-1 with density-dependence was confirmed, namely the production of antibacterial substances of bacteria NJ6-3-1 was regulated by quorum sensing. These results demonstrated sponge-associated bacteria NJ6-3-1 have chemical defense regulated by quorum sensing mechanism. On the other hand, quorum sensing signal molecules N-acyl homoserine lactones (AHLs) and diketopiperazines (DKPs), which are commonly considered to be quorum sensing signal molecules, were proved to exist in sponge-associated bacteria NJ6-3-1, and the autoinducer that regulated the metabolite of antibacterial substance in bacteria NJ6-3-1 was explored. Finally, the luxR gene fragments of quorum sensing transcriptional regulatory protein LuxR was obtained from whole genome of bacteria NJ6-3-1, thus we preliminary demonstrated that LuxI/LuxR quorum sensing regulatory system exist in bacteria NJ6-3-1 from the molecule level. This study will enrich the related theory of chemical defense induction mechanism of marine bacteria, deepen the understanding of bacteria regulation functions of quorum sensing, and provide a new way for the development of new medicinal natural products. This dissertation consists of six chapters as follow:Chapter one consists of two parts. In the first part, a brief introduction of sponge-associated microorganisms is given, including their types, secondary metabolites, and chemical defense mechanisms. In the second part, a brief introduction of bacterial quorum sensing system is summarized, and the topics discussed include the history of quorum sensing, classification of quorum sensing system and signal molecules, the formation and regulation mechanism of quorum sensing, the detection method of signal molecule AHLs, and the phenomenon of quorum sensing in marine bacteria. The research objective and experimental plan of this dissertation are also presented.In chapter two, the screening and identification of sponge-associated bacteria with antibacterial activity are discussed. At first, sponge-associated bacteria were isolated by dilution-plate method from marine organism sponge of South China Sea and East China Sea, and the antibacterial activity were screened for four pathogens as test strains by an agar diffusion method. At the same time, taxonomy was studied based on phylogenetic analysis of 16S rDNA sequences. The results showed that 19 strains (20 %) among total 95 isolated marine bacteria have antimicrobial activity. And the most active sponge-associated bacteria were identified to be the genus Bacillus.In chapter three, the antibacterial substances of sponge-associated bacteria NJ6-3-1 with cell density dependence is discussed. SectionΙandΠ, the antibacterial metabolites were isolated and purified from sponge-associated bacteria NJ6-3-1 with remarkable antibacterial activity. And combined with the chemical method and the biotechnology, two antibacterial substances in bacteria NJ6-3-1 was ultimately obtained by active tracking, the structure of two compounds was determined by many structure identification methods. The formula of the first antibacterial substance was C15H13BrO3, whose molecular weight was 320, and structure was named as 2-(1’-bromine-1’,3’-butadiene)-4-(2’’,4’’-dienevaleric acid)-phenol. It has different inhibition effect to Bacillus subtilis, Staphylococcus aureus and Saccharomyces cerevisiae, minimum inhibitory concentration (MIC) in turn was 500μg/mL, 125μg/mL and 250μg/mL. The molecular weight of the second antibacterial substance was 520, whose interim formula was C23H24BrN2SO5. It has inhibition effect to Staphylococcus aureus, MIC was 500μg/mL. SectionШ, the content of antibacterial substances C23H24BrN2SO5 of bacteria NJ6-3-1 depends on cell density was determined by simultaneous detection of the cell density of bacteria NJ6-3-1 under different culture times and the content of antibacterial substance C23H24BrN2SO5 in metabolite crude extracts of bacteria NJ6-3-1. The metabolism of antibacterial substance C23H24BrN2SO5 of bacteria NJ6-3-1 with cell density-dependence was further proved by detecting antibacterial activity of metabolite crude extracts of bacteria NJ6-3-1 under different culture times, and cell density threshold value is OD630=0.4. In chapter four, the AHLs in the metabolites of sponge-associated bacteria NJ6-3-1 is discussed. SectionΙ, the detection method of quorum sensing signal molecule AHLs in bacteria was established, and the production of AHLs in sponge-associated bacteria NJ6-3-1 was detected combined with these methods. The results showed that signal molecule AHLs which can induce biosensor strain A. tumefaciens A136 to produce blue pigment exist in bacteria NJ6-3-1, and bacteria NJ6-3-1 contains three AHLs, N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-tetradecanoyl-L-homoserine lactone (C14-HSL) by Gas Chromatography-Mass Spectrometry (GC-MS) detection. It demonstrated that this method could be applied to screen AHLs-producing bacteria from large quantities of bacteria. SectionΠ, C6-HSL, C8-HSL and C14-HSL, as quorum sensing signal molecules and the metabolites of bacteria NJ6-3-1, was added respectively in the culture of bacteria NJ6-3-1 at low cell density, and the metabolite crude extracts of bacteria NJ6-3-1 were obtained after adding different inducers. Through analyzing antibacterial activity by bioautography and the content of antibacterial substance C23H24BrN2SO5 by High Performance Liquid Chromatography (HPLC), the results showed C8-HSL can induce low-density bacteria NJ6-3-1 to produce antibacterial activity, and can induce the metabolite content of antibacterial substance C23H24BrN2SO5 of low-density bacteria NJ6-3-1 to increase, thus we concluded that C8-HSL is very likely a quorum sensing autoinducer which are produced by bacteria NJ6-3-1 to regulate the metabolite of antibacterial substance.In chapter five, the DKPs in the metabolites of sponge-associated bacteria NJ6-3-1 is discussed. At first, cyclo-(ΔVal-L-Val), cyclo-(L-Phe-L-Val) and cyclo-(L-Pro-L-Leu), as quorum sensing signal molecule and the metabolites of bacteria NJ6-3-1, was added respectively in the culture of bacteria NJ6-3-1 at low cell density, and the metabolite crude extract of bacteria NJ6-3-1 were obtained after adding different inducers. Through detecting of antibacterial activity by bioautography, the results showed cyclo-(L-Phe-L-Val) can induce low-density bacteria NJ6-3-1 to produce antibacterial activity, so it is probably that cyclo-(L-Phe-L-Val) is a quorum sensing autoinducer which are produced by bacteria NJ6-3-1 to regulate the metabolite of antibacterial substance. Secondly, 100 L broth of bacteria NJ6-3-1 were fermented in order to get more DKPs compounds from the bacteria NJ6-3-1, and twelve DKPs were isolated and purified from the metabolites of bacteria NJ6-3-1. In addition, the structure of twelve DKPs was identified by means of physical and chemical properties and spectroscopy. Furthermore, one DKPs compound, namely cyclo-(Tyr-Phe) has not yet been reported to be found in the microbial.In chapter six, the molecular basis of quorum sensing in sponge-associated bacteria NJ6-3-1 is discussed. The luxR gene fragment was sequenced. The luxR gene fragment of quorum sensing transtritional regulatory protein was found to exist in bacteria NJ6-3-1 by comparison whole genome of bacteria NJ6-3-1 (Ningbo University), thus the LuxI/LuxR quorum sensing regulatory system was initially confirmed to exist in bacteria NJ6-3-1 from genetic aspect.更多还原