【作者】 李萍；

【导师】 王磊；

【作者基本信息】 南开大学 ， 微生物学， 2013， 博士

【摘要】 2005年,诺尔曼氏极小单孢菌(Pusillimonas noertemannii)作为一个新的种属首次被提出。该属的代表株定为BN9T,这株菌作为一株5-氨基水杨酸-降解菌株,分离自一个6-氨基-2-磺酸盐-降解混合细菌培养物中。极小单孢菌T7-7(Pusillimonas sp. T7-7)是一株革兰氏阴性的,耐冷的,柴油降解菌株,该菌分离自中国渤海原油污染区域的海底淤泥中。在该采样点,一艘运油船于2002年11月23日发生了泄漏。16SrRNA基因序列分析显示T7-7与菌株Pusillimonas ginsengisoli DCY25T (98.4%序列相似度),Pusillimonas soli MJ07T(97.5%序列相似度)和Pusillimonas noertemannii BN9T(96.7%序列相似度)进化关系最为接近。而菌株T7-7与其它产碱菌科(Alcaligenaceae)已鉴定的菌种直接的16S rRNA基因序列相似度水平均低于95.3%。该结果说明菌株T7-7是极小单孢菌属中的新成员。本研究报道了T7-7的全基因组序列,这也是极小单孢菌属中报道的第一个全基因组序列。T7-7的全基因组包括了一个大约3.9M的染色质和一个大约41kb的质粒。染色质和质粒的平均GC含量分别为56.92%和56.01%。染色质共含有3,696个蛋白编码基因,2个rRNA操纵子,47个tRNA基因包括了全部20个氨基酸,以及5个假基因。质粒上共含有77个蛋白编码基因。比较基因组分析揭示了T7-7作为典型的海洋细菌的许多特性,包括缺少完整的糖代谢途径,存在完整的乙醛酸途径和糖异生途径,具有硝酸盐同化以及反硝化能力,以及硫酸盐还原和亚硫酸盐氧化等能力。T7-7可以利用柴油(C5到C30链长的烷烃)为唯一碳源和能源进行生长。烷烃通常可以以O2为电子受体进行需氧降解,或者以硝酸盐或磺酸盐为电子受体进行厌氧降解。微生物的烷烃需氧降解途径通常以O2依赖的单加氧酶作为起始,该酶是降解途径中的关键酶,它的功能是将烷烃转化为相应的烷醇。而烷醇又会被醇脱氢酶(ADH)转化为相应的醛,接着被醛脱氢酶(ALDH)转化为相应的酸,进而通过β氧化途径被进一步代谢。目前,已经有许多种类的微生物烷烃氧化酶被鉴定了,其中包括细胞色素P450烷烃单加氧酶,整合膜蛋白的非血红素离子烷烃单加氧酶AlkB以及可溶的细菌荧光素SsuD亚家族的单组份烷烃单加氧酶LadA等。由于在T7-7的基因组中没有发现烷烃降解的关键酶,如alkB,ladA或其他已知编码烷烃羟化酶的同源基因,T7-7降解柴油主要成分烷烃的能力可能涉及了新的基因。在本研究中,结合了生物信息学,蛋白组学和实时定量反转录PCR等技术,鉴定了T7-7中烷烃降解相关的基因簇。该烷烃降解系统由一个Rieske家族的单加氧酶,一个铁氧还蛋白和一个NADH依赖的还原酶组成。该单加氧酶包括了一个46.711kDa的大亚基和一个15.355kDa的小亚基,它们的功能通过体外的理化实验和体内的异源功能重组实验等被进一步证实。纯化后的单加氧酶大亚基可以以NADH为辅酶,氧化链长在正戊烷(C5)到正二十四烷(C24)之间的烷烃,在以正十五烷(C15)为底物时获得最大活性。存在铁氧还蛋白及NADH依赖的还原酶时,该活性可以被增强。该单加氧酶大亚基在以包括硝基甲烷,甲磺酸等几种烷烃衍生物为底物时,也表现了一定的活性,而以芳香烃化合物为底物时则无活性。该单加氧酶大亚基的最适反应条件为pH7.5,温度30℃。Fe2+可以明显增强该酶的活性。这是首次在细菌中发现了一个属于Rieske非血红素离子氧化还原酶家族的烷烃单加氧酶系统。与其它已鉴定的烷烃氧化酶相比,T7-7中的烷烃单加氧酶系统显示了更加优越的耐低温特性,在接近0℃时仍能保持活性。这种特性使其具有应用在更多低温环境下的催化进程中的潜能。更多还原

【Abstract】 In2005, the creation of a new genus and species with the name Pusillimonas noertemannii gen. nov., sp. nov. is proposed. The type strain is the Pseudomonas-like strain, designated BN9, which was isolated as a5-aminosalicylate-degrading organism from a6-aminonaphthalene-2-sulphonate-degrading mixed bacterial culture.Pusillimonas sp. T7-7is a Gram-negative cold-tolerant diesel oil-degrading bacterium isolated from the seabed mud of a petroleum-contaminated site in Bohai Sea, China. At this location, an oil tanker leaked on23November2002. Partial16S rRNA gene sequence analysis indicated that this strain was related most closely to Pusillimonas ginsengisoli DCY25T (98.4%sequence similarity), Pusillimonas soli MJ07T(97.5%) and Pusillimonas noertemannii BN9T (96.7%). The levels of16S rRNA gene sequence similarity between strain T7-7and other recognized species of the family Alcaligenaceae were below95.3%. This suggested that strain T7-7represented a member of the genus Pusillimonas.We present here the complete genome sequence of T7-7. It is the first complete genome sequence of the genus Pusillimonas. The complete genome of T7-7contains a chromosome of about3.9M and a plasmid of about41kb. The average GC content is56.92%for the chromosome and56.01%for the plasmid. The chromosome contains3,696protein-encoding genes,2rRNA operons,47tRNA genes for all20amino acids, and5pseudogenes. The plasmid has77protein-encoding genes. Comparative genome analysis revealed many features of typical marine bacteria, including the absence of intact sugar metabolic pathways, the presence of glyoxylate and gluconeogenesis pathways, and the abilities for nitrate assimilation and denitrification, as well as sulfate reduction and sulfite oxidation.Pusillimonas sp. T7-7is able to utilize diesel oils (C5to C30alkanes) as a sole carbon and energy source. Alkanes may be degraded aerobically with O2or anaerobically using nitrate or sulfate as the electron acceptor. Microbial aerobic alkane biodegradation is usually initiated by O2-dependent monooxygenases, the key enzyme of the degradation pathway and this converts alkanes to corresponding alkylalcohols. The alkylalcohol is converted to alkylaldehyde by alcohol dehydrogenase (ADH), and then to fatty acids by aldehyde dehydrogenase (ALDH) to be further degraded via the β-oxidation pathway. Several types of microbial alkane oxygenases have been characterized, including cytochrome P450alkane monooxygenases, the integral membrane non-heme iron alkane monooxygenases, such as AlkB, and the soluble bacterial luciferase SsuD subfamily single-component alkane monooxygenase LadA etc. As no homologues of alkB, ladA, or other known genes encoding alkane hydroxylase, the key enzyme of alkane degradation, were found in T7-7, the ability to degrade alkanes, which are the major components of diesel oils, seems to involve novel genes.In this present study, using a combination of bioinformatics, proteomics and real-time reverse-transcriptase polymerase chain reaction (RT-PCR) approaches, the alkane degradation gene cluster in Pusillimonas sp. T7-7was identified. This system is composed of a Rieske-type monooxygenase, a ferredoxin and a NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711kDa) and one small (15.355kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15substrate. The activity can be enhanced by the presence of ferredoxin and the NADH-dependent reductase. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optima reaction condition of the large subunit is pH7.5at30℃. Fe2+can enhance the activity of the enzyme evidently.This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium. Compared to other characterized alkane oxygenases, the Pusillimonas sp. T7-7 alkane monooxygenase system shows better cold-tolerance, with activity retained at temperatures as low as0℃. This property makes it an excellent candidate to be used in various catalytic processes where low temperature conditions are encountered.更多还原