摘要
群体感应是一种普遍存在的微生物细胞与细胞之间的信息交流机制。群体感应使单细胞微生物之间能够在“群体水平”上“相互协作”,完成一些单细胞无法进行的生物过程。以酰基化高丝氨酸内酯(Acylated Homoserine Lactone, AHL)作为信号分子的群体感应系统广泛存在于革兰氏阴性细菌中,并在许多生物过程中都扮演了重要角色。
微生物是污染物生物降解的主力军,但是群体感应在污染物生物降解过程中的作用缺乏研究。仅一篇文献报道AHL信号分子的添加能够改变活性污泥的微生物菌群组成,并提高活性污泥污染物处理能力的稳定性。但AHL信号分子是否能在污染物降解过程中合成,群体感应对微生物的生物降解能力的调控及机制尚未见报道。本学位论文以芳香族化合物作为典型的难清除有毒污染物,以AHL介导的群体感应作为典型研究对象来探讨群体感应在污染物降解过程中的作用。这将为我们从微生物群体视角来认识污染物生物降解的调控过程提供有用信息,同时也对从群体角度开发新的高效生物降解策略有着重要意义。
本论文首先拟以AHL群体感应系统和芳香族污染物生物降解过程作为典型对象建立一个简单的研究模型。通过筛选得到了一株具有AHL群体感应系统且有较强芳香族污染物降解能力的铜绿假单胞菌(Pseudomonas aeruginosa) CGMCC 1.860,并对其降解特性进行了分析。PCR及生物信息学分析表明,该菌苯酚羟化酶基因簇由一个转录因子(phcR)和六个亚基(phcPONMLK)组成,而儿茶酚间位途径基因在一个由转录因子(nahR)和十个结构基因组成的NAH基因簇上,关键酶儿茶酚2,3-双加氧酶(C230)由nahH编码。另一方面,分析确认了该菌具有完整的Rhl群体感应系统(rhlIR基因簇),并在LB培养基中具有短链AHL合成能力。进一步通过同框缺失(In-frame deletion)技术,构建了Rhl群体感应系统的缺失菌株△rhlI,△rhlIR。该简单模型以P. aeruginosa CGMCC1.860为供试菌株,芳香族污染物为底物,可用于研究Rhl群体感应系统对芳香族污染物生物降解的调控作用。
AHL信号分子定量检测方法非常缺乏,尤其是对有毒环境样品中AHL信号分子的定量检测方法至今未见报道。本研究通过筛选得到了一株能够在BHL(N-butyryl-homoserine lactone,一种AHL,Rhl群体感应系统的信号分子)添加条件下产生绿色色素(pyocyanin)的铜绿假单胞菌,通过基因工程手段将该菌株构建成了一个以色素产生为信号输出的BHL定量检测生物传感器,利用该传感器可以简单地通过分光光度法对BHL进行定量检测。其最低检测限较以往的方法有大幅降低。同时对该生物传感器的抗干扰能力、稳定性、可靠性进行了验证,并且在污水样品中得到了成功的应用。该方法为进一步深入研究群体感应信号分子在污染物降解过程中的积累规律及群体感应对污染物降解过程的调控机制提供了简单经济的定量检测工具。
虽然已报道从活性污泥等污染物处理过程中分离到了能够在复合培养基中产生AHL信号分子的菌株,但是至今未有报道表明AHL信号分子能否在污染物生物降解过程中合成。同时关于群体感应对微生物污染物降解能力的影响作用至今未见报道。通过生物传感器检测、薄层层析(TLC)和HPLC-APCI-MS/MS分析表明P. aeruginosa CGMCC1.860在芳香族污染物降解过程中能够合成BHL(N-butyryl homoserine lactone)和HHL(N-hexanoyl-homoserine lactone),且BHL为主要成分。接着,我们发现P. aeruginosa CGMCC 1.860的AHL粗提物能够提高该菌的苯酚降解能力,但不含有短链AHL的粗提物对该菌的苯酚降解能力无明显影响。该结果表明外源添加的短链AHL(主要是BHL,由rhlI合成)能够提高铜绿假单胞菌的苯酚降解能力。由此推测Rhl群体感应系统在该过程中对生物降解具有调控作用。通过比较野生型菌株、△rhlI、△rhlIR及其回补菌株的降解能力,结果表明Rhl群体感应系统能够提高铜绿假单胞菌的芳香族污染物降解能力。
我们进一步探索了Rhl群体感应系统对芳香族污染物降解关键酶酶活及基因转录水平的影响。以苯酚降解作为典型有毒芳香族污染物生物降解过程,通过对三种条件下(野生型,△rhlI,△rhlI+BHL)菌株降解过程的比较分析表明,Rhl群体感应系统能够在转录水平上正向调控转录因子nahR表达,以nahH为代表的NAH基因簇也受到Rhl群体感应系统的正向调控,同时C230酶活也受到群体感应的正向调节。由此推测,Rhl群体感应系统对儿茶酚间位途径的促进是其提高芳香族污染物降解能力的主要原因。另一方面,在对数生长后期,Rhl群体感应系统对苯酚羟化酶系统phcR和phcP,全局性调控因子relA, spoT, crc的表达都表现出负调控。这些结果显示了群体感应在污染物降解过程对细胞调控的复杂性,同时也为进一步深入研究提供了基础。
本研究建立了一种用于环境样品中AHL信号分子定量检测的生物传感器,证明了AHL信号分子能够在污染物降解过程中合成,并发现Rhl群体感应系统能够提高铜绿假单胞菌芳香族污染物降解能力,进一步获得了群体感应系统对芳香族污染物调控的分子信息。这些信息和工具为今后进一步深入研究群体感应对芳香族污染物生物降解的调控机制,从群体视角有目的地调节芳香族污染物的生物降解,奠定了良好的基础,并对于研究其他群体感应系统在其他污染物降解过程中的作用也具有良好的借鉴作用。
Quorum sensing (QS) is a widely conserved cell-to-cell communication system in micro-organisms especially for bacteria. This QS process enables bacteria to cooperatively control behaviour at population level and, therefore, effectively complete the processes which are unproductive when undertaken by an individual bacterium. Acylated homoserine lactone (AHL) mediated QS is wide-spread in Gram-negative bacteria and plays important roles in many biological processes.
Biodegradation by micro-organisms especially bacteria is crucial for pollutant treatment. But, the role of QS in pollutants biodegradation is still unclear. There is only one related report which indicated AHL addition could change the microbial community and improve the stability of activated aludge. But no information about AHL production or QS role in pollutants biodegradation was available. In this work, the role of QS in pollutant biodegradation was investigated in a model system. This work provided us important information to re-visit the regulation process in pollutants biodegradation from a population perspective and, also is useful for effective biodegradation strategy development based on this new perspective.
At first, a simple model system based on aromatic pollutants biodegradation and AHL QS system was developed. Pseudomonas aeruginosa CGMCC 1.860 was screened out due to the presence of AHL QS system and its good aromatics biodegradation ability. Further analysis of its aromatics biodegradation was also conducted. In addition, the Rhl QS system (rhlIR cluster) was confirmed in this strain and it could synthesis short-chain AHL in LB medium as confirmed by biosensor assay. This developed model system could be useful for investigation of the regulation on aromatics biodegradation by Rhl QS system. Furthermore, mutants of△rhlI,△rhlIR were constructed by gene in-frame deletion and, therefore facilitate to unveil the RhI regulation at molecular level.
Quantification method for AHL is still severely limited. Until now, there is no report about method for AHL quantification in toxic environmental sample. By genetic modification, an isolated aromatic degrader was constructed to be a BHL biosensor. By using the pigment production which responded to exogenous BHL as biosensor output, BHL quantification in samples was simply achieved with spectrophotometric detection. The sensitivity for BHL detection was improved by using this developed biosensor compared with previous methods. In addition, the biosensor output showed nice interferences resistance, good stability and reliability. It was also successfully applied to environmental samples even without extraction. The developed method is useful for quantitative investigation of the role of QS signals in environmental processes.
Recently, strains which can synthesis AHL in rich medium were isolated from pollutants treatment processes. However, there is still no evidence to indicate AHL could be synthesized during pollutants biodegradation and no report about the effect of QS system on pollutants biodegradation ability of bacteria. By biosensor detection, TLC and HPLC-APCI-MS/MS analysis, BHL and HHL were identified during aromatics biodegradation. In addition, AHL crude extract addition improved phenol biodegradation by P. aeruginosa CGMCC 1.860. But crude extract without short chain AHL did not significantly affect its biodegradation ability. The results indicated that AHL could enhance phenol biodegradation by P. aeruginosa. In addition, the aromatics degradation ability for△rhlI and△rhlIR was significantly reduced compared with the wild type strain. By BHL addition in△rhlI or plasmid complementation in△rhlIR, their degradation ability could be restored. The results strongly indicated Rhl QS system could improve the aromatics biodegradation ability of P. aeruginosa.
Furthermore, the regulation by Rhl QS system on aromatics biodegradation was studied at enzyme and transcription level. Using phenol biodegradation as a typical example, the transcription level of nahR and nahH was up-regulated by Rhl QS system. The activity of catechol 2,3-dioxygense was also positively regulated by Rhl QS system. The results suggested Rhl QS system could activate the catechol meta-cleavage pathway and therefore improved the phenol biodegradation by P. aeruginosa CGMCC 1.860. Interestingly, our results indicated that the transcription level of phenol hydroxylase gene cluster (phcR, phcP) and global regulators (relA, spoT, crc) was down-regulated by Rhl QS system at late logarithmic phase. This finding provided important information for the mechanism investigation.
In this work, a biosensing system for BHL quantification in environmental sample was developed; AHL production during aromatics biodegradation was identified; the regulation of Rhl QS system on aromatics was investigated; The information and tool provide a good basis for further investigation of QS regulation on aromatics biodegradation. This study is also considered useful for manipulation aromatics biodegradation at population perspective. The idea may be useful for investigation of regulation on other pollutants biodegradation by other QS systems.
引文
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