【作者】 王蕾；

【导师】 陶美凤； 张承才；

【作者基本信息】 华中农业大学 ， 微生物学， 2007， 博士

【摘要】 大肠杆菌的ftsK是同时参与新生横隔的形成以及染色体分离的细胞分裂必需基因。天蓝色链霉菌在整个生活周期中并没有标准的细胞分裂过程，但是基因组中却存在ftsK的同系物(ftsK_SC)。本研究旨在探索ftsK_SC在链霉菌形态分化中的功能。首先在天蓝色链霉菌中构建了ftsK_SC的两个缺失突变株：一个ftsK_SC的读码框被acc（3）Ⅳ-oriT基因盒置换得到WL1，另一个则将ftsK_SC进行同框缺失得到WL11。表型观察和互补实验证明ftsK_SC的缺失突变导致链霉菌遗传不稳定。生物信息分析预测FtsK_SC同FtsK／SpoⅢE家族大多数成员一样由相对独立的跨膜结构域的N末端、具有保守的依赖ATP的DNA转运酶结构域的C末端以及中间的连接区组成。而FtsK_SC主要结构域的同框缺失蛋白均不能实现功能互补，表明这三个主要结构域都是FtsK_SC维持链霉菌遗传稳定性所必需的。预萌发实验、氯霉素敏感突变以及精氨酸营养缺陷型突变的检测，表明ftsK_SC缺失突变引发了染色体不稳定。5个PCR扩增实验结果表明大部分的异常菌落中存在染色体一个或两个末端片段的丢失，而且丢失的最大片段可能超过841 kb。FtsK_SC-EGFP融合蛋白在链霉菌菌丝体中的定位观察结果表明，FtsK_SC主要作用于链霉菌产孢时期，跨膜结构域是正确的细胞定位以及维持遗传稳定所必需的。利用sigFp：egfp作为报告基因，发现ftsK_SC缺失突变导致约7％的孢子虽然含有DNA但无法正常表达egfp。对这些孢子类似物的观察发现在孢子类似物中sigF的表达是自主调节的，同时也暗示了未知的产孢晚期基因位于由FtsK_SC负责转运的染色体的末端片段上。观察ftsK_SC缺失突变株中FtsZ_SC-EGFP融合蛋白的细胞定位，发现天蓝色链霉菌的FtsK_SC缺失突变没有影响到细胞分裂蛋白FtsZ_SC在气生菌丝中的定位。同样作为链霉菌染色体的分离蛋白，ParAB和FtsK_SC的双突变株中未观察到明显的突变累计效应，表明ParAB和FtsK_SC在链霉菌产孢时期的染色体分离过程中的作用可能是相对独立的。S1核酸酶作图发现ftsK_SC至少有两个启动子，一个位于ftsK_SC的起始密码子上游400 bp左右，另一个位于osaB的上游，暗示着ftsK_SC与osaB存在共转录。多个启动子的存在也暗示了ftsK_SC的表达可能存在较严谨的调控。最后构建了基因组中另一个ftsK同系物SCO4508的多个缺失突变株，均未观察到明显的表型变化。生物信息分析显示该基因可能是YukA类转运蛋白，是革兰氏阳性细菌中第四类分泌系统的重要组成蛋白。更多还原

【Abstract】 Streptomyces coelicolor A3（2） does not have a canonical cell division cycle duringmost of its complex life cycle, yet it contains a gene (ftsK_SC) encoding a protein similar toFtsK, which couples the completion of cell division and chromosome segregation inunicellular bacteria such as Escherichia coli. This work is to investigate the role of ftsK_SCduring the development of Streptomyces.Two ftsK_SC null mutants were constructed: WL1, containing an aac（3）Ⅳgenereplacement, and WL11, an in-frame deletion mutant. The observation and complementexperiments demonstrated that the ftsK_SC null mutation resulted in genetic instability inStreptomyces.Bioinformatic analysis indicated that, like other FtsK/SpoⅢE family members, FtsK_SCconsists of three main parts: an N-terminal trans-membrane domain was separated from aC-terminal conserved ATP-dependent DNA translocase domain by a linker region. Thecomplement experiments of truncated FtsK_SC versions suggested that the three parts areall important for the maintenance of genetic stability.The germination experiment and the increased frequency of thechloramphenicol-sensitive and arginine auxotrophs mutation indicated that the ftsK_SCdeletion might be associated with genomic instability. The results of 5 PCR experimentsshowed that most of abnormal colonies had lost one or both of the chromosome terminalfragments and some of these deletions removed more than 841 kb.The observation of the FtsK_SC-EGFP fusion protein located in Streptomyces hyphaeindicated a role for FtsK_SC in chromosome segregation during sporulation. It alsosuggested that the cytoplasmic location of FtsK_SC is required for its function.Use of a fluorescent reporter showed that in ftsK_SC mutans several spore compartmentsin most spore chains failed to express the late sporulation-specific sigma factor gene sigF,even though they contained chromosomal DNA. The observation of these sporecompartments suggested that sigF expression is autonomously activated in each sporecompartment in response to completion of chromosome transfer which would thus be apreviously unknown checkpoint for late sporulation-specific gene expression.The location of the FtsZ_SC-EGFP fusion protein in the fisK_SC null mutant showed that inS. coelicolor the ftsK_SC deletion didn’t affect the location of the division protein FtsZ_SCduring sporulation. None of the mutant cumulative effect was been observed in the parAB and ftsK_SCdouble mutants implying that the roles of ParAB and FtsK_SC in chromosome segregationare substantive.S1 nuclease protection revealed that there were at least two promoters to transcriptftsK_SC: one was over 400 bp upstream the start coden GTG; the other was upstream theosaB gene implying co-transcription. More than one promoter for ftsK_SC implies that theexpression of ftsK_SC may be regulated in a strict and complicated way.Another ftsK-related gene SCO4508 in S. coelicolor was disrupted. No distinctphenotype was observed compared with the parent strains respectively. Bioinformaticsanalysis of functional domains suggested that the SCO4508 product may be a YukA-liketransporter protein, a crucial component of the Gram-positive typeⅣsecretion system.更多还原