基于基因组和差异蛋白质组学分析的北冰洋沉积物中Pseudomonas fragi胞壁聚酯合成初步研究

Polar marine bacterium Pseudomonas fragi A22 was isolated from the sediment of the Arctic Ocean located in the Arctic Circle. Granular polyester of monolayer (named A22B) was formed around the cell wall of the strain. The A22B extracted from the strain A22 has these properties: sudan black staining positive but water soluble, retained in a dialysis tubing with molecular weight of 8000 daltons, and no detectable sugar or amino acid residues in its hydrolysates. Both morphological and chemical analysis indicated that A22B was different from the PHB produced by Pseudomonas sp., capsule of Azotobacter vinelandii, lipopolysaccharide from Escherichia coli and protease liquid beads from P. fragi B25. The morphological properties, distribution of the site, coloring, product analysis after hydrolysis indicated that the A22B was a novel hydrophilic polyester without glycosyl group. The key problem is that neither literature about the polymer nor the genomics and proteomics of biocontrol bacteria can be referred to this day. The synthetic mechanisms and metabolic regulation of A22B are encountering a bottlenecks. Comparative genomics and proteomics are efficient methods to explore the genetic basis for the formation of A22B in the strain. This project firstly plans to obtain the whole genomes of the P. fragi A22 and the type strain P. fragi B25 (ATCC4973) by high-throughout sequencing, assembling and genomic walking filling the gap. Then, genes function of the genomes for A22 and B25 will be predicted, annotated and analyzed. Next, the different information of their genomes are analyzed by comparative genomics in order to discuss the relationship between the formation of A22B around the A22 strains and the genetic background. The differential proteomics between A22 and B25, between A22 and its A22B deletion mutants obtained by transpositional recombination will be analyzed, respectively. Furthermore, the properties, function and the associated metabolic pathway of the specific proteins will be studied. This research aims to fill the genome blank of the Pseudomonas fragi in the database, explain the genomic difference between A22 and B25, clarify the differential proteomics between A22 and B25, between A22 and its A22B deletion mutant, reveal their nature, function and associated metabolic pathway, preliminarily find the synthetic pathways associated with A22B or functional gene based on genomics and proteomics. These study will lay an important theoretical foundation for the settlement of the metabolic regulation of A22B from the strain A22.

从位于北极圈内的北冰洋沉积物中分离到极地海洋细菌莓实假单胞菌 A22，该菌在细胞壁形成单层排列的高分子聚酯（简称A22B）。定性分析表明：A22B 是一种不含糖环、单一母核的亲水性新型酯类高分子聚合物。目前存在的问题是A22B 的形态和结构新颖，无相关高分子物质文献和模式菌基因组和蛋白质组信息参考，A22B 的合成机制等研究遇到瓶颈问题。本项目首先对菌株A22 和模式菌B25进行全基因组测序和比较基因组学分析，探讨A22菌株形成高分子聚酯的遗传背景；然后，分析A22 与模式菌、A22与其A22B缺失突变株的生理变化、蛋白表达谱差异及其关联的代谢途径。本研究成果旨在填补模式菌基因组在数据库中的空白，阐明A22 与B25、A22 与其A22B缺失突变株的生理变化及差异蛋白表达谱，揭示其性质、功能及相关的代谢途径，发现A22B合成的特有途径或功能基因，为进一步探讨A22B 代谢调控奠定基础。

耐冷微生物是生物技术开发的宝库，莓实假单胞菌是极地耐冷细菌，在其细胞壁周围分泌一种特殊的细菌外膜囊泡。该菌由全长5.1-Mb的基因组及三个质粒组成，比较基因组分析发现该菌株特有的代谢途径。获得了六种新的次级代谢产物合成基因簇，分析了其特有的单芳环碳氢化合物，包括羟基苯、羟基联酚、和羟胺, 聚芳环化合物等代谢途径，这些途径说明该菌具有降解环境污染物相关基因。此菌还具有独有的碳固定途径，可能有助于P.fragi在恶劣环境条件下的生存，还存在6-巯基嘌呤、氟尿嘧啶降解代谢途径，这很可能是一种自身的脱毒保护系统。分析了基因岛和转运蛋白的多样性，含有不饱和脂肪酸合成相关基因，进一步说明该菌能够适应冷环境的遗传背景。该菌具有Ⅳ型分泌系统，说明了其外膜不同于其他假单胞菌的典型特征，极地嗜冷菌可能在碳、氮、磷、等的循环和固定等方面有其重要作用。通过Label free蛋白质组学非标记定量技术，分别对P.fragi 4℃和20℃培养的全细胞蛋白质组学研究结果发现，共有130种蛋白质表达存在差异，另有257种蛋白质存在有无差异表达。低温条件下，与RNA合成、加工及结构调控的相关蛋白, 及一些转录元件上调表达, 说明转录和翻译过程参与细菌的低温适应；参与丙酮酸代谢的酶类含量显著上升，同时增加不饱和脂肪酸的合成，以维持低温下细胞膜的流动性；随着温度的下降, 细菌细胞内ATP和整个腺苷酸库水平都会提高，与能量代谢相关的酶类含量也有显著上升。通常认为，外膜囊泡是细菌对外界环境的一种应急响应，本项目通过超高速离心法获得了P.fragi外膜囊泡、初步验证了其对抗生素的耐受特点，分析了外膜囊泡主要蛋白质组成及脂多糖PE和PG，说明了外膜囊泡是一种能够包裹蛋白质及脂类物质的一种复合体。外膜囊泡很可能是生命世界与非生命世界物质交换的桥梁。本项研究为后续外膜囊泡功能开发、合成机制的深入研究奠定理论基础，为次级代谢产物沉默基因簇的表达及耐冷酶的开发提供数据支持。

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