群体感应信号分子对不动杆菌降解石油烃的调控机制

Acinetobacter is a genus of Gram-negative bacteria capable of degrading crude oil and widely distributed in nature. Many studies have focused on the investigation of the biodegradation mechanisms in Acinetobacter species on the individual level. However, few data have been presented about the mechanisms on the ‘muticelluar’ level. Bacteria behave as single cellular organisms at low cell densities, however, they may shift from a unicellular to a largely multicellular existence and modify gene expression levels to initiate a broad range of group behaviors that benefit the growing community. They use small molecule or peptidic signals to assess their local population densities in a process termed quorum sensing (QS). It has been suggested that the autoinducer(AI), QS signal molecules play important roles in biofilm formation. Disruption of the gene encoding for QS signals such as acyl homoserine lactone (AHLs) could not result in biofilm development-mediated cell-cell communications. .One bacterial strain which was originally isolated from marine sediments was identified to be affiliated with the genus Acinetobacter and named Acinetobacter sp. strain HC8-3 in our laboratory. The strain HC8-3 was found to be able to degrade crude oil efficiently and could form biofilm during the biodegradation process. The Lux operon gene cluster was also discovered from the genomic data. Therefore, the question that ‘How the QS system may control complex processes including biofilm formation and biodegradation of hydrocarbons?’ is raised. To this end, 1) the QS signal molecules will be extracted and identified using TLC, GC-MS, 2) the hydrocarbon-degrading and biofilm-formation capabilities of the wild type bacterial strain and mutants will be compared along with the proteomic data and genomic data. Proteomics analysis performed by ITRAQ technique will reveal the differentially expressed proteins in the bacterial strain which are regulated by QS system. The QS system involved in the regulation of factors that influence the formation of biofilms and growth on hydrocarbons will be analyzed accordingly. A better understanding of the specific QS signals that regulate biofilm formation will contribute to more efficient biodegradation in pollutant-contaminated environments..The insights and theories of this study will enhance our understanding of the regulatory functions of quorum sensing on the biodegradation process. It will also provide fundamental evidences for in situ bioremediation of petroleum hydrocarbon pollutions in the marine system.

不动杆菌属细菌是广泛存在的石油烃降解功能菌。对该属细菌的个体代谢途径研究较多，而对其群体代谢途径的研究尚欠缺。申请人前期工作中分离得到一株来源于海洋的不动杆菌（Acinetobacter sp. HC8-3），能够降解石油烃，形成生物膜，具有群体感应系统相关基因。基于此，本项目提出“群体感应系统对HC8-3形成生物膜及降解石油烃的调控机制是怎样的”这一科学问题。拟通过信号分子检测及降解效率测定，研究HC8-3信号分子特征与功能；通过全基因组分析及突变菌株构建，揭示群体感应系统调控机制；并基于差异蛋白质组分析，阐明群体感应系统对生物膜形成及石油烃降解功能的影响。从而揭示HC8-3群体感应系统对生物膜形成及石油烃降解的调控机制。这一研究将丰富我们对群体感应系统在石油烃降解生物过程中的认识，为揭示细菌降解石油烃的分子机制提供理论依据，对构建海洋石油烃污染的生物修复技术具有重要参考价值。

本研究针对高效石油烃降解菌Acinetobacter sp. HC8-3S，通过全基因组测序分析、突变菌株构建和差异蛋白质组学分析，阐明群体感应系统对石油烃降解功能的调控机制。研究结果发现：1）通过报告平板法和气相色谱-质谱法检测，Acinetobacter sp. HC8-3S能够产生N-酰基高丝氨酸内酯类群体感应信号分子，并且添加此类信号分子能够显著提高HC8-3S菌株对正十六烷的降解效率；2）全基因组分析显示，HC8-3S菌株中具有4类HTH-LUXR结构域的群体感应信号分子合成基因（1193、1192、401和1614）；3）利用同源重组方法，成功构建了群体感应系统基因缺失突变株△1193。该突变株丧失了对正十六烷的降解作用，生物膜形成能力显著降低，并影响烷烃羟化酶编码基因alkM的转录；4）差异蛋白质组学研究发现，石油诱导前后，突变株与野生株相比，受到影响的生物过程主要为脂肪酸代谢过程、脂质代谢过程、脂肪酸β氧化过程、脂质分解代谢过程、氧化还原过程等。香叶醇、亮氨酸、异亮氨酸、已内酰胺、柠檬烯、蒎烯等的代谢途径发生了显著变化。本研究丰富了我们对群体感应系统在石油烃降解生物过程中的认识，为揭示群体感应与石油烃降解的耦合作用机制提供理论依据，对建立石油污染的生物修复技术具有重要的参考价值。

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