两篇文章解析TubZ分离系统的分离与调控机制

　　

　 近期，中国科学院武汉病毒研究所袁志明研究员课题组在TubZ质粒分离系统的分离及其调控机制的研究中取得重要进展，相关结果发表在国际微生物学刊物Microbiology-SGM 和Journal of Bacteriology上。

　　TubZ质粒分离系统（plasmid partition system）是近年来被发现的一类新的分离系统（III型分离系统），它编码DNA结合蛋白TubR和自组装蛋白TubZ。该系统目前只在蜡样芽胞杆菌群的pBtoxis和pXO1质粒上被发现，目前还没有相关稳定因子及其调控机理的报道。

　　球型芽胞杆菌由于能够产生高毒力的杀蚊二元毒素蛋白而被广泛应用于疾病媒介蚊虫的生物防治。质粒pBsph是该实验室首次在球形芽胞杆菌C3-41中发现的一个大质粒（178 kb）。该研究发现，质粒pBsph上一个2.3 kb的DNA片段组成了一个最小复制子，它包含有tubZ和tubR基因以及上下游部分DNA片段。TubR与上游DNA区域重复序列（tubC）的结合不仅为质粒分离所必需，而且能够在转录水平上负调控分离操纵子的表达。TubZ能与TubRC结合形成三元核蛋白复合体TubZRC，在质粒的分离和稳定中发挥重要作用。

   通过进一步研究，课题组首次发现了TubZ系统一个新的调控因子TubX，它能够和tubRZ操纵子的启动子结合。TubX能够和TubR竞争结合DNA，通过拮抗TubR的负调控活性来正调控分离蛋白表达，进而参与质粒的分离和稳定。基于上述结果，该课题组首次提出了TubZ分离系统的一个调控模型，这对于进一步理解TubZ介导的遗传物质的稳定遗传机制及二元毒素的进化具有十分重要的意义。

　　该研究得到了国家重点基础研究发展计划（“973”计划）和国家自然科学基金的支持。

原文摘要：

A new tubRZ operon involved in the maintenance of the Bacillus sphaericus mosquitocidal plasmid pBsph.
pBsph is a mosquitocidal plasmid first identified from Bacillus sphaericus, encoding binary toxins (Bin toxins) that are highly toxic to mosquito larvae. This plasmid plays an important role in the maintenance and evolution of the bin genes in B. sphaericus. However, little is known about its replication and partitioning. Here, we identified a 2.4 kb minimal replicon of pBsph plasmid that contained an operon encoding TubR-Bs and TubZ-Bs, each of which was shown to be required for plasmid replication. TubR-Bs was shown to be a transcriptional repressor of tubRZ-Bs genes and could bind cooperatively to the replication origin of eleven 12 bp degenerate repeats in three blocks, and this binding was essential for plasmid replication. TubZ-Bs exhibited GTPase activities and interacted with TubR-Bs : DNA complex to form a ternary nucleoprotein apparatus. Electron and fluorescence microscopy revealed that TubZ-Bs assembled filaments both in vitro and in vivo, and two point mutations in TubZ-Bs (T114A and Y260A) that severely impaired the GTPase and polymerization activities were found to be defective for plasmid maintenance. Further investigation demonstrated that overproduction of TubZ-Bs-GFP or its mutant forms significantly reduced the stability of pBsph. Taken together, these results suggested that TubR-Bs and TubZ-Bs are involved in the replication and probably in the partitioning of pBsph plasmid, increasing our understanding of the genetic particularity of TubZ systems.

A novel transcriptional activator tubX is required for the stability of Bacillus sphaericus mosquitocidal plasmid pBsph.
Stable maintenance of the low-copy-number plasmid pBsph in Bacillus sphaericus requires partitioning (par) system that consists of a filament-forming protein TubZ-Bs, a centromere-binding protein TubR-Bs, and a centromere-like DNA site tubC, composed of three blocks (I, II and III) of 12-bp degenerate repeats. Previous studies have shown that mini-pBsph replicons encoding TubZ system are segregationally highly unstable, whereas the native pBsph is stably maintained. However, the mechanism underlying the stability discrepancy between pBsph and its minireplicon is poorly understood. Here, a downstream gene orf187 (encoding TubX) of tubZ-Bs was found to play a role in plasmid stabilization. Null mutation or overexpression of tubX resulted in a defect in pBsph stability and a significant decrease in tubRZ-Bs expression, and the TubX-minus phenotype was suppressed by ectopic expression of a wild-type copy of tubX and additional tubRZ-Bs. Electrophoresis mobility shift assay (EMSA) and DNase I footprinting revealed a direct binding of TubX protein to five 8-bp degenerate repeats located in the par promoter region, and that TubX competed for binding to the par promoter with TubR-Bs. Further studies demonstrated that TubX significantly stimulated transcription of the par operon in the absence of tubR-Bs, and a greater magnitude of gene activation was observed when tubR-Bs was present. These results suggested that TubX positively regulates the tubRZ-Bs transcription by interfering with TubR-Bs-mediated repression and binding directly to the tubRZ-Bs promoter region.