【作者】 张俊杰；

【导师】 周宁一；

【作者基本信息】 中国科学院研究生院（武汉病毒研究所） ， 微生物学， 2006， 博士

【摘要】 硝基芳香烃化合物是重要的化工原料，常作为医药、染料、农药等合成的前体。这类化合物能在环境中长期残留，已经对土壤、水体和大气造成了严重的污染。硝基芳香烃化合物能影响人体的物质代谢，有致癌、致畸和致突变等危险，严重威胁着人类的健康和生存。苯环结构的对称性和稳定性使硝基芳香烃化合物不易发生化学反应，而环境中的许多微生物却能够利用这些污染物作为唯一的碳源、氮源和能源来生长，能把这些污染物完全降解。微生物的这一特性使其在污染物的生物治理和生物修复中具有难以替代的优势，因而越来越受到人们的青睐。同时，对这些微生物的代谢途径和代谢基因的研究也成为本领域研究的热点。本论文的研究工作分为以下三个部分。第一部分是Pseudomonas sp．WBC-3对4-硝基酚的代谢途径研究。WBC-3是一株能够利用甲基对硫磷(Methyl Parathion，MP)和4-硝基酚(4-Nitrophenol，4-NP or PNP)做为唯一碳源、氮源及能源生长的革兰氏阴性菌。WBC-3降解MP的第一步是在甲基对硫磷水解酶(Methyl parathionhydrolase，MPH)的作用下生成4-硝基酚，然后再由其它的酶系完成4-硝基酚的代谢。目前报道的4-硝基酚的代谢途径主要有两种，根据其中间代谢产物的不同分别称之为对苯二酚途径和1，2，4-苯三酚途径。对苯二酚途径主要存在于在革兰氏阴性菌中，而1，2，4-苯三酚途径则在革兰氏阳性菌中广泛存在。我们前期的研究结果表明革兰氏阴性菌WBC-3是经对苯二酚途径降解4-硝基酚。在该途径中，4-硝基酚首先被氧化为对苯二醌(Benzoquinone)，对苯二醌在还原酶作用下还原为对苯二酚(Hydroquinone)，然后进入下面的开环途径。对苯二酚在对苯二酚双加氧酶的作用下开环生成γ-羟基粘康酸半醛(γ-Hydroxymuconic semialdehyde，HMSA)，后者在脱氢酶作用下转化为马来酰乙酸(Maleylacetate，MA)。MA在MA还原酶作用下生成β-酮己二酸(β-ketoadipate)并最终进入三羧酸循环。目前唯一的与4-硝基酚代谢相关的基因及操纵子的报道来自于革兰氏阳性菌Rhodococcus opacus SAO101(Kitagawa et al，J Bacteriol，2004：4894)，该菌经1，2，4-苯三酚途径降解4-硝基酚。目前尚没有关于4-硝基酚经对苯二酚途径代谢的分子生物学研究的文献报道。本研究通过基因组步移的方法，成功地获得了4-硝基酚经对苯二酚途径代谢的完整的基因簇。序列比对结果表明，在14 kb长的片断中可能包括了参与4-硝基酚代谢的全部基因。其中pnpA编码4-硝基酚单加氧酶，pnpB编码对苯二醌还原酶，pnpC编码对苯二酚双加氧酶，而γ-羟基粘康酸半醛脱氢酶和马来酰乙酸还原酶分别由pnpD和pnpE编码。WBC-3的代谢基因簇可能属于3个不同的操纵子，其中pnpA和pnpB反向转录，负责上游代谢途径，pnpCDE组成下游代谢操纵子，在它们的上游有一个可能编码LysR家族调控蛋白的基因。本研究通过体外表达和基因敲除鉴定了WBC-3代谢4-硝基酚途径中的两个关键酶基因，即催化初始反应的4-硝基酚单加氧酶(para-nitrophenol monooxygenase，PNPMO)和催化开环反应的对苯二酚双加氧酶(Hydroquinone dioxygenase，HQDO)。pnpA编码的4-硝基酚单加氧酶能够在体外催化4-硝基酚氧化为对苯二酚并同时释放等当量的亚硝酸根离子。序列分析结果表明，4-硝基酚单加氧酶属于典型的黄素蛋白单加氧酶家族，它与现有的所有功能类似的单加氧酶没有明显的亲缘关系，在进化过程中形成一个独立的分支。pnpC编码的对苯二酚双加氧酶也是WBC-3降解4-硝基酚的必需基因，序列比对及进化分析结果表明该酶属于苯三酚1，2-双加氧酶家族。对4-硝基酚单加氧酶的酶学特性的研究结果表明，该酶的酶活需要FAD和NADPH作为辅因子，每转化一个分子的4-硝基酚需要消耗两个分子的NADPH。该酶底物特异性很强，仅能催化4-硝基酚和4-硝基邻苯二酚的氧化反应，其中4-硝基酚是该酶的最适底物。本研究是首次在分子生物学水平上研究4-硝基酚的对苯二酚代谢途径和代谢机理。与R．opacus SAO101的苯三酚代谢途径相比，WBC-3无论是代谢途径，酶和基因，还是代谢操纵子的结构特点都与之完全不同。第二部分是Pseudomonas sp．WBC-3对芳香烃化合物的趋化特性研究。微生物的趋化是指微生物个体朝向或者远离某种刺激物的运动，是对外界环境变化作出的迅速的反应。根据趋化与微生物代谢的关系可以将微生物的趋化分为代谢依赖型趋化和代谢不依赖型趋化。对于降解有机污染物的微生物来讲，趋化可能是细菌进化产生的一种选择优势，但是细菌的趋化特性及其在生物降解和生物修复中的作用目前仍没有引起足够的重视。本研究采用drop assay方法研究WBC-3的趋化特性，结果表明，WBC-3不仅能够对其可代谢物4-硝基酚、4-硝基邻苯二酚和对苯二酚产生趋化反应，而且对多种其不能降解或转化的芳香烃化合物也能产生趋化反应。此外，WBC-3的趋化是组成型表达的，是不依赖于代谢的趋化，代谢基因的敲除不影响它的趋化反应。WBC-3对芳香烃化合物的趋化可能属于β-ketoadipate控制的趋化系统，这种趋化系统在降解有机污染物的微生物中广泛存在，是在进化过程中微生物获得的一种选择优势。第三部分是Pseudomonas sp．WBC-3的降解质粒pZWLO的部分基因组生物信息学分析和注释。质粒作为可移动遗传元件(Mobile Genetic Elements，MGEs)的一种在微生物对环境污染物的适应性进化中起着关键的作用，许多代谢途径的基因簇都定位在质粒上。目前已有多个降解质粒的全基因组序列测定完成，通过对其进行比较分析不仅能直接获得代谢基因簇的全序列，而且还可以用来研究代谢基因的起源和进化，并为质粒的复制调控机理及其进化研究提供了材料。Pseudomonas sp．WBC-3中的甲基对硫磷水解酶(MPH)基因就定位在一个约70kb的质粒(pZWLO)上，本研究对已获得的该质粒的基因组序列进行分析和注释。结果表明，在已获得的序列中，主要是与质粒的复制、稳定传代和接合转移有关的核心基因。从复制蛋白的序列来看，质粒pZWLO可能属于IncP不相容组，但具体属于哪个不相容亚组还不明确。pZWL0中质粒核心基因的氨基酸序列与数据库中同类蛋白的序列一致性都不高，这说明pZWL0与现有的已知序列的降解质粒在进化中的亲缘关系较远。序列分析结果表明WBC-3的甲基对硫磷水解酶基因(mph)定位于一个典型的Ⅰ型转座子上(Tn-mph1)，而该转座子又是Ⅱ型转座子(Tn-mph2)的一部分。目前已知的两类有机磷水解酶(MPH和OPD)基因都定位在转座子上，这可能是它们在微生物中广泛存在的原因。进化分析结果表明二者起源于不同的祖先，经过独立的进化过程形成两种结构完全不同的同工酶。更多还原

【Abstract】 Nitroaromatic compounds are common starting materials for the synthesis ofcomplex industrial N-containing compounds and can be major environmentalcontaminants, hazardous metabolic intermediates or dead-end products.Microorganisms play an important role in transforming these recalcitrantcontaminants and in the associated recycling of the nitrogen. In the pastdecades, the intensive research in this area has led to dramatic progress inunderstanding the microbial strategies and associated mechanisms for thedegradation of nitroaromatic compounds. A combination of increasingcommercial interest and advances in our understanding of the genetic andbiochemical basis of biodegradation is expected to produce a more rationalapproach to bioremediation technology.This dissertation is composed of three parts.The first part is determination of the molecular basis for 4-nitrophenoldegradation in Pseudomonas sp.strain WBC-3.WBC-3 can utilize methyl parathion (MP) and 4-nitrophenol (4-NP) as the solesource of carbon, nitrogen and energy. The key enzyme which catalyses theinitial reaction in MP degradation of WBC-3 is methyl parathion hydrolase(MPH), which converts MP to 4-NP stoichiometrically.To date, several 4-NP degrading bacteria have been isolated and theirdegradation pathways have been studied in details. The degradation pathway inwhich 4-NP is converted to maleylacetate via hydroquinone (hydroquinonepathway) was preferentially found in Gram-negative bacteria. The degradationpathway in which 4-NP is converted via 4-nitrocathechol (4-NCA) andhydroxyquinol (hydroxyquinol pathway) was preferentially found inGram-positive bacteria.The results of this study indicate that Pseudomonas sp. strain WBC-3 degraded4-NP through the typical hydroquinone pathway proposed by Spain inMoraxella (Spain et al, Appli.Environ.Microbiol., 1991: 812). The pathway proceeds through oxidative elimination of the nitro group to form benzoquinonewhich is subsequently reduced to hydroquinone in the presence of NADPH. Thelatter was cleavaged resulting in the formation ofγ-hydroxymuconicsemialdehyde by a soluble enzyme. Upon addition of catalytic amounts ofNAD~+,γ-hydroxymuconic semialdehyde was converted to maleylacetic acid,which enters the tricarboxylic acid cycle through theβ-ketoadipic acidpathway.Although many studies of 4-NP degradation have been reported, geneticinformation on 4-NP degradation remains limited and the only characterized4-NP degradation gene cluster originated from a Gram-positive bacterium,Rhodococcus opacus SAO101, which degradated 4-NP via hydroxyquinolpathway. No genes encoding degradation of 4-NP through hydroquinonepathway has been reported so far. Therefore, it was of great interest to cloneand characterize the catabolic genes involved in 4-NP metabolism to verify thehydroquinone pathway proposed by Spain. In particular, functionalcharacterization of the 4-nitrophenol monooxygenase, the enzyme involoved inthe initial reaction of the pathway, is highly desirable.In this report, a novel 4-NP degradation gene cluster from Gram-negativebacterium, Pseudomonas sp. WBC-3, was identified and characterized. First, abenzoquinone reductase gene, which is presumed to play a role in convertingbenzoquinone to hydroquinone in 4-NP degradation, was amplified by PCRbased on the conserved sequences. Second, the flanking regions of this genefragment were cloned and sequenced by genomic walking strategy. Finally, atotal of 14 kb contig was generated by assembling the sequence acquired fromthree genomic walking reactions. Fourteen possible ORFs were identified inthis contig and annotations of these ORFs were completed based on the resultsof BLAST analyses. Of these ORFs, the deduced amino acid sequence of PnpA,which was proposed to encode the para nitrophenol monooxygenase(PNPMO),shows moderate homology (25% identity) with pentachlorophenol4-monooxygenase of Sphingobium chlorophenolicum ATCC 39723 and pnpCappears to encode the dioxygenase for the cleavage of hydroquinone. Genesencoding benzoquinone reductase (pnpB),γ-hydroxymuconic semialdehydedehydrogenase (pnpD) and maleylacetic acid reductase (pnpE) were alsoidentified. Analyses of the sequenced region demonstrates that the genes involoved in thedegradation of 4-NP in WBC-3 were arranged in at least three operons, pnpBand pnpA are adjacent to each other but transcribed in opposite directions whilethe genes encoding the lower pathway of 4-NP degradation, pnpCDE, arenearby in operonic association with several open reading frames coding forproteins of unknown function. A putative regultor was also found upstream ofpnpCDE operon. Sequence alignment and phylogenetic anaylis indicate PnpArepresents a new member of FAD monooygenases and PnpC is a close relativeto hydroxyquinol 1, 2-dioxygenase family.In order to confirm the function of the putative genes, pnpA and pnpC wereexpressed in E.coli respectively. Conversion of 4-NP to hydroquinone wasobserved when expressed pnpA in E.coli with concomitant release of nitrite.Furthermore, either pnpA or pnpC deleted strain completely lost the ability togrow on 4-NP as sole carbon source. These results clearly suggested that bothpnpA and pnpC play essential roles in 4-NP mineralization in strain WBC-3.Characterization of PnpA as 4-nitrophenol monoxygenase was also conductedin the current study. PnpA demonstrates a narrow substrate range and E. colicells expressing pnpA can only attack 4-nitrophenol or 4-nitrocatechol. Theenzyme activity requires both FAD and NADPH as cofactors. NADH can serveas electron donor for the reaction but was less effective as NADPH.Interestingly, the reaction in crude extract required 2 mol of NADPH for theoxidation of each mol of 4-nitrophenol. These data suggest that the initialmonooxygenase converted 4-nitrophenol into benzoquinone at the expense of 1mol NADPH. The benzoquinone was then reduced to hydroquinone bybenzoquinone reductase at the expense of an additional mol of NADPH.The second part of this dissertation focuses on the chemotaxis features ofPseudomonas sp. strain WBC-3 toward aromatic compounds.The drop assay was used to show that multiple aromatic compounds arechemoattractants for Pseudomonas sp. strain WBC-3. Among thesechemoattractants, only 4-nitrophenol, 4-nitrocatechol and hydroquinone can bedegraded or transformed by WBC-3. The results presented here clearlysuggested that the chemotactic response of WBC-3 is constitutive and ismetabolism-independent based on the fact that (1) non-metabolizablecompounds are also attractants for WBC-3 and (2) the catabolic mutant strains do not affect its chemotaxis behavior. It was suggested that strain WBC-3 has aβ-ketoadipate chemotaxis system that responds to a wide range of aromaticcompounds, which is similar to that present in Pseudomonas PRS2000, themodel organism for chemotaxis study. The broad specificity of this chemotaxissystem works as advantage in WBC-3 and other soil bacterial because itsfunctions to dectect diverse carbon sources.The third part of this dissertation is the annotation of DNA sequence from thecatabolic plasmid pZWL0 of Pseudomonas sp. WBC-3.pZWL0 carrys the catabolic gene mph (methyl parathion hydrolase), which isresponsible for the conversion of methyl parathion to 4-nitrophenol inPseudomonas sp WBC-3. Analysis of the partial complete nucleotide sequence(76 kb) revealed 62 potential opening reading frames (ORFs) and most of thoseare related to the core functions of plasmid such as replication, stablemaintenance and transfer. The plasmid maybe attribute to the IncPincompatibility group based on the identities of the putative replicon protein ofpZWL0. However, the sequence and organization of the backbone genes ofpZWL0 show lower identities with known plasmids, which may imply itsdistant relationships with other catabolic plasmids.The sequence analysis also reveal that the mph gene in WBC-3 is flanked bytwo copies of insertion sequence (IS6100), which is the typical character ofclassⅠtransposon. This transposon, designated Tn-mph1, is a composition of alarge transposon, designated Tn-mph2, which is a classⅡtransposon accordingto its sequence and organization characters.To data, two types of organophosphate hydrolase had been identified, oneis MPH-like and the other is OPD-like. Genes encoding both enzymes areproposed to be physically located in a typical transposon, which may beresponsible for the widely distribution of these enzymes. However, these twokinds of enzymes share poor sequence identities and phylogenetic analysissuggested that organophosphate hydrolases were multioriginate and MPH,which evolved independently from OPD, diverged fromβ-lactamase family.更多还原