根瘤菌 Azorhizobium caulinodans ORS571的染色體分配蛋白質ParA 與 ParB之功能探討

Functional characterization of the ParA and ParB chromosome partition proteins in Azorhizobium calinodans ORS571

彭敏華(Min-Hua Peng 彭敏華)

指導教授 ： 劉啟德

共同指導教授 ： 李昆達(Kung-Ta Lee)

國立臺灣大學/生命科學院/生化科技學系/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10487

摘要

根瘤菌會與豆科植物共生形成根瘤， 而整個結瘤過程牽涉到植物與微生物間複雜的訊息交換。初期的根瘤形成與後期表達固氮作用的訊息傳導因子已經被陸續發現。相較於此，中期的根瘤成熟階段，也就是根瘤菌在植物細胞內經由分化作用形成類菌體（bacteroid）到表達共生固氮前所牽涉的分子訊息傳達機制，至今依然不明。我們先前的研究發現，Azorhizobium caulinodans ORS571 根瘤菌的染色體分配系統（ParA 與 ParB 蛋白）除了參與染色體分配以外，也在類菌體分化過程中扮演著負調控者的角色。本研究利用 Azorhizobium 根瘤菌的 ParA 與 ParB 重組蛋白探討染色體分配系統與類菌體形成關連基因間的交互作用。 ParA 蛋白屬於 Walker type ATPase，當添加 ParB 時會促進 ParA 的ATP 水解活性。ParB 蛋白因具有 H-T-H DNA binding domain，可與 centromere-like 的 parS 基因結合，而添加 ParA 會增進上述結合能力。在 in vitro co-immunoprecipitation 實驗發現， ParA 與 ParB 彼此間有交互作用。 根據 EMSA (Electrophoretic mobility shift assay) 分析的結果， ParB 不僅會和一個與類菌體形成有直接關連的基因 (bacA) 結合，也會與 par 操作組的啟動子結合。此外，本研究也發現 ParA/ ParB 的莫耳數比例會影響
ParB 對於 bacA 基因的結合能力。綜合本研究與先前研究的結果推論，A. caulinodans 根瘤菌的類菌體分化調控，是透過 ParA 與 ParB 之間的交互作用，進而影響類菌體形成相關基因的轉錄活性。

關鍵字

類菌體分化 ； Azorhizobium caulinodans ORS571根瘤菌 ； 染色體分配蛋白

並列摘要

The nodulation process of legume-rhizobium symbiosis is highly complex, involving a succession of interactions between the host plants and the microsymbionts. However, little is known about the molecular basis of events in nodule maturation process, i.e. after the bacteria are released from infection threads into the nodules and differentiating into bacteroid before nitrogen fixation begins. Our precious experimental evidence has demonstrated that the azorhizobial chromosome partition system (ParABS) not only played crucial roles in the partitioning of chromosome, but also negatively regulated the bacteroid formation process in nodulation. In this study, the functions of the azorhizobial recombinant ParA and ParB proteins, and the interactions between the par system and bacteroid differentiation related genes have been analyzed. ParA have been regarded as a Walker-type ATPase, and its ATP hydrolysis activity was enhanced in the presence of ParB protein. The ParB contains a helix-turn-helix (HTH) motif which is responsible for interaction with DNA. In the electrophoretic mobility shift assay (EMSA), ParB bound to a centromere-like parS DNA fragment of A. caulinodans, and this binding was stimulated by the increasing amounts of ParA protein. Interactions between ParA and ParB were confirmed by in vitro immunoprecipitation assay. In the electrophoretic mobility shift assay (EMSA), a bacteroid differentiation associative gene (bacA) or the promoter region of par operon was allowed to bind the ParB protein. and the ratio of ParB to ParA seems to be the key factor to regulate this binding. A mechanism for regulation of bacteroid differentiation of A. caulinodans was proposed. Interactions between azorhizobial ParA and ParB proteins may function as a checkpoint, which couples the chromosome partitioning to the onset of bacteroid formation.

並列關鍵字

Bacteroid differentiation ； Azorhizobium caulinodans ORS571 ； ParA ； ParB

參考文獻

Altschul, S. F., Thomas, L. M., Alejandro, A. S., Jinghui, Z., Webb, M. &David, J. L. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.

Austin, S. &Abeles, A. (1983). Partition of unit-copy miniplasmids to daughter cells. II. The partition region of miniplasmid P1 encodes an essential protein and a centromere-like site at which it acts. J Mol Biol 169: 373-387.

Austin, S. &Wierzbicki, A. (1983). Two mini-F-encoded proteins are essential for equipartition. Plasmid 10: 73-81.

Autret, S., Nair, R. &Errington, J. (2001). Genetic analysis of the chromosome segregation protein Spo0J of Bacillus subtilis: evidence for separate domains involved in DNA binding and interactions with Soj protein. Mol Microbiol 41: 743-755.

Bartosik, A. A., Lasocki, K., Mierzejewska, J., Thomas, C. M. &Jagura-Burdzy, G. (2004). ParB of Pseudomonas aeruginosa: interactions with its partner ParA and its target parS and specific effects on bacterial growth. J Bacteriol 186: 6983-6998.