【作者】 骆迎峰；

【导师】 于军；

【作者基本信息】 浙江大学 ， 生物学， 2009， 博士

【摘要】 Phenylobacterium zucineum strain HLK1~T是我们从肿瘤细胞k562细胞系中分离得到的兼性细胞内共生细菌。P.zucineum可以侵染并稳定生长繁殖于宿主细胞,但不影响宿主细胞生长和形态。基于16S rDNA的系统发育分析表明P.zucineum细菌属于alpha变形菌纲Phenylobacterium属。P.zucineum是Phenylobacterium属唯一具有真核细胞侵染能力的成员。为从基因组角度了解该兼性内共生细菌的基本代谢、和强环境适应能力等特征,我们对P.zucineum进行了全基因组测序及序列分析。P.zucineum包含一个长为3,996,255 bp的环状染色体和一个长为382,976 bp的质粒;共有3,861个蛋白编码基因,42个tRNA基因和一个16S-23S-5S rDNA操纵子。P.zucineum基因组编码大量双组分信号传导蛋白,转录调节因子,外周作用sigma因子和热激反应相关基因。P.zucineum基因组所含双组分信号传导蛋白和转录调节因子的相对数目在同类兼性细菌中最高,而转录调节因子数目甚至超过了环境适应能力最强的广生境细菌的平均水平。在我们所分析的占据代表性系统进化地位并具有代表性生活方式的93种细菌中,P.zucineum含有数目最多的热激反应相关基因。比较基因组学分析表明双组分信号传导蛋白和转录调节因子含量与细菌环境适应能力密切相关。所以P.zucineum基因组编码的高数量环境应激相关基因可能是其具有高环境适应能力的重要原因。蛋白谱同源性比较和16S rDNA系统进化分析同时表明原核生物细胞周期研究模式生物Caulobacter crescentus是与P.zucineum最相似的全基因组序列测序细菌。C.crescentus和P.zucineum基因组比较发现两细菌约含60%直系同源基因,不存在明显的基因组共线性。我们还发现大量环境应激相关基因分布在P.zucineum特异质粒上。进化分析表明基因水平转移和质粒内基因重复在塑造P.zucineum质粒编码环境适应蛋白谱过程中发挥重要作用。研究表明CtrA,GcrA,DnaA和双相信号传导蛋白在C.crescentus细胞周期调控过程中发挥重要作用。P.zucineum和C.crescentus基因组比较分析表明两者的细胞周期调控机制高度保守。具体说来,P.zucineum和C.crescentus ctrA基因含有完全一致的启动子结构和DNA甲基转移酶识别位点。P.zucineum含有参与C.crescentus ctrA基因转录,CtrA蛋白磷酸化和降解过程相关基因的全部直系同源基因。P.zucineum和C.crescentus CtrA靶基因保守性远大于两基因组平均水平,并且含有结构相似的CtrA结合位点。此外,我们还发现参与复制、重组或修复的GcrA靶基因和DnaA靶基因,以及影响细胞周期进行、细菌生长或形态发生的双相信号传导蛋白基因在P.zucineum和C.crescentus基因组中相对保守。本论文主要展示了Phenylobacterium属第一个全基因组测序细菌的基因组分析结果。基因组注释有利于鉴定P.zucineum丙苯氨酸利用、细胞内生存、高环境适应性等生理特征的遗传基础。比较基因组学分析表明P.zucineum具有类似于原核生物细胞周期研究模式细菌Caulobacter crescentus的细胞周期调控机制。更多还原

【Abstract】 Phenylobacterium zucineum strain HLK1~T is a facultative intracellular species recently isolated from the human leukemia cell line K562.P.zucineum maintains a stable association with its host cell without affecting the growth and morphology of the latter. Phylogenetic analysis based on 16S rDNA revealed that P.zucineum belongs to the genus Phenylobacterium.P.zucineum was the only bacterium reported to invade eukaryotic cells in the genus Phenylobacterium.To better understand the basic metabolism and high capacity of environmental adaptation of this facultative intracellular bacterium from genomic perspective,we carry out the whole genome sequencing of P.zucineum.This work would present the main findings on the genomic analysis of P.zucineum.The P.zucineum genome consists of a circular chromosome(3,996,255 bp) and a circular plasmid(382,976 bp).It encodes 3,861 putative proteins,42 tRNAs,and a 16S-23S-5S rDNA operon.P.zucineum genome encodes a large number of two-component transduction systems protein,transcriptional regulator,extracytoplasmic function sigma factors and heat-shock related proteins.Further comparative genome analysis indicated that P.zucineum has the highest fraction of two-component transduction proteins among common facultative bacteria and even has the fraction of transcriptional regulator higher than mean level of multiple bacteria which are well accepted as bacteria with the highest capacity of environmental adaptation.Notably,P.zucineum genome harbors the largest number of heat-shock related proteins among analyzed 93 bacteria which occupy the major phylogenic positions and live in the major representative environmental niches.Therefore,the high number of environmental stress proteins encoded in P.zucineum genome may partial account for its high capacity of environmental transition and adaptation.Both phylogenetic analyses based on 16S rDNA gene and comparative protein profiles analysis indicated that P.zucineum is phylogenetically closest to Caulobacter crescentus,a model species for bacterial cell cycle research.Comparative genomic analysis indicated that about 60%of P.zucineum protein-coding genes have C.crescentus orthologs.There is no apparent synteny between the C.crescentus chromosome and P. zucineum countpart.We also found most of P.zucineum specific stress genes are located in plasmid.Both horizontal gene transfer and gene duplication within plasmid play important roles in the shaping of P.zucineum plasmid stress proteins profiles.Researches have revealed CtrA,GcrA,DnaA and two-component transduction proteins play important roles in C.crescentus cell cycle regulation.Comparative analysis indicated cell cycle regulation system is well conserved between P.zucineum and C. crescentus.Detailly,P.zucineu and C.crescentus ctrA gene have identical promoters(p1 and p2) and the DNA methyltransferase(CcrM) recognization motif(GAnTC).P.zucineu has all the orthologs participating in C.crescentus ctrA gene transcription,CtrA phosphorylation and proteolytic degradation.The fraction of CtrA-regulated genes with orthologs in P.zucineum is significantly greater than the mean level of the whole genome. P.zucineum and C.crescentus CtrA-regulated genes have structure similar CtrA binding sites.Potential GcrA-regulated genes and DnaA-regulated genes involving in replication, recombination or repair are well conserved between P.zucineum and C.crescentus two-component transduction genes required for cell cycle progression,growth or morphogenesis are more conserved than that function in stress environment.This work presents the first complete bacterial genomic analysis in the genus Phenylobacterium.Genome annotation facilitates us to identify the genetic basis of P. zirconium’s physiological features,such as phenylalanine utilization,intracellular living style and the high capacity of environmental adaptation.Comparative genomic analysis indicated that the cell cycle regulation systems are well conserved between P.zucineum and Caulobacter crescentus,a well known cell cycle research model prokaryote.更多还原