【作者】 张巨源；

【导师】 张承才；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 博士

【摘要】 蓝细菌是地球上最为古老的物种之一。一些丝状蓝细菌在缺氮环境中,菌丝体上有部分细胞会分化成专门执行固氮功能的异形胞（heterocyst）。化石证据和地质学证据表明,异形胞大约出现在24亿年前,是地球上已知的最早出现的细胞分化现象之一。异形胞形成机制是蓝细菌研究的重要领域。最近二十多年来,发现了近百个与异形胞发育密切相关的基因（如氮代谢过程中的全局调控基因ntcA,异形胞分化中心调控基因hetR,固氮酶基因nifHDK,异形胞胞被合成基因hepA、hglE等）和一些小分子信号(如2-OG、Ca²⁺等)。这些基因和信号分子构成了一个复杂的异形胞分化调控网络。然而该调控网络在进化中是如何形成的却仍不清楚,与此相关的研究也还处于空白。在本论文中,我们用比较基因组学的方法研究了异形胞发育的进化过程。我们检测了产异形胞蓝细菌Anabaena PCC7120中已知的异形胞发育相关基因（het基因）在其他18种蓝细菌基因组中的分布,发现大部分的het基因还存在于其它不产异形胞菌株的基因组中。根据分布情况,het基因可分为以下几类:一,在多数蓝细菌中都存在的基因;二,只在丝状菌株中存在的基因;三,只在固氮菌株中存在的基因;四,主要在产异形胞菌株中存在的基因;五,其他分布类型的基因。利用分布情况和序列信息,我们分析了每个het基因的进化过程。结果表明,一,het基因出现在蓝细菌进化的各个阶段;二,绝大部分的het基因在异形胞分化现象出现之前就已经存在于蓝细菌基因中了;三,水平基因转移在异形胞进化过程中发挥了重要作用。在Anabaena PCC7120中,HetR和PatS共同调控了异形胞分化起始以及其在菌丝上的分布。我们在被检测的几种不产异形胞的丝状蓝细菌中皆发现了HetR的表达,并在已知基因组序列的几种丝状蓝细菌（包括不产异形胞菌株TrichodesmiumIMS101和Arthrospira platensis的基因组）中皆发现了patS基因。Arthrospira platensis细胞内的HetR蛋白在缺氮后表达增加。在Anabaena PCC7120中Arthrospiraplatensis的hetR基因（arhetR）会刺激异形胞分化,而Arthrospira platensis的patS基因（arpatS）会抑制异形胞分化,并且它们也分别受到与Anabaena自身hetR和patS类似的转录调控。此结果表明,hetR和pats基因在产异形胞蓝细菌和不产异形胞蓝细菌的共同祖先基因组中就已存在了,并且那时它们就已形成了相互作用的方式。基于上述的生物信息分析和实验工作,我们推测了异形胞发育的进化过程。在异形胞出现之前,许多重要的异形胞发育调控因子之间就已经形成了一个基本氮利用调控网络,异形胞的进化实质上就是一个不断获取必需的het基因,并将其纳入该网络中的过程。从根本上说,异形胞的产生是蓝细菌为了更好利用氮源而不断适应环境的结果。本研究结果将有助于了解生物界最初的演化过程,也可以为研究高等生物的细胞分化提供启示。我们在螺旋蓝细菌基因组中发现了长度为957b bp的基因,其编码序列与酵母和植物中的3’,5’-二磷酸腺苷-5’-磷酸酶（PAPase）很相似,该基因被命名为halA。PAPase是硫同化途径中必需的一个酶,它负责将硫同化过程中产生的毒性副产物3’,5’-二磷酸腺苷-5’-磷酸（PAP）转化为无毒性的AMP。在真核生物中已经发现了多个PAPase,它们对Li⁺/Na⁺都非常敏感,极低浓度的Li⁺/Na⁺就可使其失去活性,从而引起细胞内PAP的积累,最终产生细胞毒性。作为细胞内对盐离子最敏感的酶之一,PAPase的耐盐能力往往决定了细胞的耐盐能力。我们将halA基因在E.coli中进行了克隆、表达和生化性质研究。酶活测定表明HalA是一种Mg²⁺依赖型的PAPase,它对PAP和3’-phosphoadenosine-5’-phosphosulfate（PAPS）皆具有特异性的降解活性。比起大多数的PAPase,HalA对Li⁺和Na⁺具有优良的耐受性(Li⁺和Na⁺对HalA活性的50%抑制浓度[IC₅₀]分别为3.6 mmol/L和600 mmol/L)。结构分析显示,HalA与Hal2p蛋白在底物结合位点存在一定差异,此差异很可能使得HalA蛋白表现出更好的Li⁺/Na⁺耐受性,并对PAP具有较弱的结合力。表达了HalA的大肠杆菌在含高浓度Li⁺的培养基中生长情况明显比对照好,意味着原核生物中PAPase可能同样是盐离子毒性的靶点。由于HalA是已知对Na⁺具有最不敏感的PAPase,因此在其他生物中表达HalA将可能是有效提高生物耐盐能力的途径之一。以前关于PAPase的研究工作都仅限于真核生物,我们在此首次对原核生物的PAPase进行了深入研究。此研究结果不仅可为了解原核生物硫代谢具体过程提供有用信息,也可为将PAPase进行改造以提高生物的硫同化效率及抗盐能力提供依据。更多还原

【Abstract】 Cyanobactera are among the most ancient organisms on the Earth.Some species of filamentous cyanobacteria can fix the atmosphere N₂ into ammonium in heterocysts,cells differentiated form vegetative cells upon nitrogen deficiency.Heterocyst differentiation is thought to having originated between 2,450 and 2,100 Ma based on geological data and fossil records,and thus it could be one of the earliest developmental phenomena.The mechanism of heterocyst differentiation is an attractive subject in the research areas of cyanobacteria.In recent years,many important genes involved in heterocyst development（het genes） have been identified,such as ntcA（global regulator in nitrogen metabolism）,hetR（regulator for initiation of heterocyst differentiation）,nifHDK （nitrogenase）,hepA and hglE（required for the formation of heterocyst envelope）,etc. Some small moleculars such as 2-oxoglutarate and calcium,are also shown to be important signals for heterocyst development.We have known much about the mechanism of heterocyst differentiation;however,how did heterocyst evolve during evolution?Here we studied the evolution history of the regulatory network involved in heterocyst differentiation by comparative genomics.Many het genes in Anabaena PCC7120 were found to be present in the genome of other cyanobacteria as well, including those non-heterocystous strains.According to their distribution,het genes can be assigned into several catalogs:1) genes in most strains,2) genes exclusively in filamentous strains,3) genes exclusively in diazotrophic strains,4) genes exclusively in heterocystous strains;5) other genes.The sequence information and distribution of het genes were used to rebuild their evolutionary history.The result shows that 1) het genes were obtained during the whole evolution history of cyanobacteria,2) most of het genes appeared before the occurrence of heterocyst,3) lateral gene transfer（LGT） played very important roles in the evolution of heterocyst development.In heterocystous cyanobacterium Anabaena PCC7120,HetR and PatS interact and act as the central regulators for the initiation of heterocyst differentiation and location of heterocysts in filaments.The hetR gene was found to be present in all examined filamentous cyanobacteria.We detected the expression of HetR in each tested filamentous cyanobacterium.We also found the ORF of the patS gene in five genomes of filamentous cyanobacteria（including non-heterocystous strains Trichodesmium IMS101 and Arthrospira platensis）.The expression of HetR in Arthrospira platensis increased upon nitrogen deficiency.The hetR gene and patS gene from Arthropira platensis （arhetR and arpatS,respectively） stimulated and repressed heterocyst differentiation when expressed in Anabaena PCC7120,respectively.The results showed that arhetR and arpatS could regulate heterocyst development in heterocystous strains.Combining the bioinformatie analysis and experimental work in this study,we reconstructed the evolution history of the regulatory network for heterocyst development. A basic network for nitrogen metabolism consisting of many key regulators for heterocyst development probably existed much earlier before the occurrence of heterocyst,and the process of heterocyst evolution is actually a process of recruiting more and more het genes to perfect this network,for effectively utilizing nitrogen nutrition in changing environments.Our results will provide clues to the ancient events in life evolution,and be helpful for understanding the developmental mechanism in higher eukaryotic organisms.3’-Phosphoadenosine-5’-phosphatase（PAPase） is required for the removal of toxic 3’-phosphoadenosine-5’-phosphate（PAP） produced during sulfur assimilation in various eukaryotie organisms.This enzyme is a well-known as the target of lithium and sodium toxicity and as the limiting factor of salt-resistance of its host.One gene,halA,which could encode a protein closely related to the PAPases of yeasts and plants,was identified from the cyanobacterium Arthrospira（Spirulina） platensis.Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades,suggesting multiple origins of PAPases in cyanobacteria.The HalA polypeptide from A.platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties.HalA was dependent on Mg²⁺ for its activity and could use PAP or 3’-phosphoadenosine-5’-phospbosulfate （PAPS） as a substrate.HalA is sensitive to Li⁺(50%inhibitory concentration[IC₅₀]≈3.6 mmol/L) but only slightly sensitive to Na⁺(IC₅₀≈600 mmol/L).The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts,which are much more sensitive to both Li⁺ and Na⁺.Based on the structure of Hal2p,the PAPase in yeast,we modeled the structure of HalA.The major difference between them was in their substrate-binding sites.The putative substrate-binding motif of HalA has a proline residue（P254） which might lower its affinity with PAP and contribute its tolerance to Li⁺ and Na⁺.The E.coli strain expressing halA gene grew much better in the medium with high[Li⁺],suggesting PAPase also was the target of lithium and sodium toxicity in bacteria as well.HalA is the known PAPase that has best tolerance to Na⁺,so it is a potential way to improve the sodium-tolerance of various organisms by expressing halA gene.Here we identified and characterized HalA,a PApase from prokaryote for the first time.The results will help us to understand the sulfur-assimilating pathway and the mechanism of sodium-resistance in bacteria.更多还原